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Am J Physiol Heart Circ Physiol 283: H2177-H2186, 2002; doi:10.1152/ajpheart.00605.2001
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Vol. 283, Issue 6, H2177-H2186, December 2002

SPECIAL TOPICS
Inhibitors of gap junctions attenuate myogenic tone in cerebral arteries

Guy Lagaud1,2, Venkateswarlu Karicheti2, Harm. J. Knot3, George J. Christ2, and Ismail Laher1

1 Department of Pharmacology and Therapeutics, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada; 2 Institute for Smooth Muscle Biology, Department of Urology and Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461; and 3 Department of Pharmacology and Therapeutics, University of Florida, Gainesville, Florida 32610


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The effects of two structurally distinct inhibitors of gap junction communication were studied by using three different forms of vasoconstriction in pressurized rat middle cerebral arteries. The sensitivity of myogenic tone (at 60 mmHg), vasopressin-induced tone (10 nM, at 20 mmHg), and depolarizing solution-induced tone (80 mM K+, at 20 mmHg) to inhibition by heptanol (1.0 µM to 3.0 mM) or 18alpha -glycyrrhetinic acid (18alpha -GA, 1.0 to 50 µM) were determined. Pressure-induced myogenic tone was inhibited by heptanol (IC50 = 0.75 ± 0.09 mM) and 18alpha -GA (~30 µM). Vasopressin-induced vasoconstriction was also inhibited by heptanol (IC50 = 0.4 ± 0.3 mM) and 18alpha -GA (>1 µM). Depolarizing solution-induced vasoconstriction was less sensitive to inhibition by heptanol compared to vasopressin (P < 0.01) or pressure-induced constriction (P < 0.05). However, 18alpha -GA did not inhibit depolarization-induced constriction. Sharp microelectrode experiments on isolated arteries revealed stable membrane potentials, with no detectable effect of heptanol (1 mM) or 18alpha -GA (20-30 µM) on the average membrane potential at 20 mmHg. However, approx 20% of impaled cells (5 of 28) exhibited uncharacteristic oscillations in membrane potential after pharmacological uncoupling. At 60 mmHg a approx 7- to 9-mV hyperpolarization and corresponding vasodilation (approx 50%) was observed, and the frequency of membrane potential oscillations doubled (9 of 23 cells). These data indicate that gap junctions play an important role in the maintenance and modulation of membrane potential and tone in cerebral resistance arteries.

heptanol; glycyrrhetinic acid; vascular smooth muscle; resistance arteries; vasopressin and depolarization


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

RESISTANCE ARTERIES react to increases and decreases in transmural pressure by constriction and dilation, respectively. This ability to respond to pressure in a manner independent of neurohormonal modulation (15) resides in vascular smooth muscle cells and was therefore termed a "myogenic response" (22). Currently, the mechanisms producing pressure-induced constriction are complex and not fully understood. However, elevations in pressure produce smooth muscle depolarization, which coincides with arterial constriction, and in some studies the production of spontaneous action potentials (20-21, 24). This in turn results in an inward movement of Ca2+ via voltage-operated Ca2+ channels. The net result of Ca2+ influx (associated with membrane depolarization), coupled with enhanced myofilament calcium sensitivity, is an increase in vascular tone (15, 24, 28). Despite the stable arterial diameter commonly observed in small pressurized cerebral arteries, recent studies in resistance arteries demonstrate great individual variability in Ca2+-signaling modalities in individual myocytes within the vascular wall, suggesting a more complicated level of signaling (33, 45, 48). The mechanistic basis for the observed and seemingly coordinated myogenic response, presumably via the propagation of myocyte depolarization and synchronization of constriction among individual myocytes, along the length of the artery is not fully understood. Our hypothesis is that one such possible mechanism is intercellular communication through gap junctions (4, 7, 14).

Gap junctions are channels formed by the docking of two hemichannels across the extracellular space of adjacent myocytes. Each hemichannel is a hexameric structure of identical (homomeric) or nonidentical (heteromeric) gap junction proteins referred to as connexins. The mammalian connexins comprise a gene family, with at least 15 members ranging in molecular mass from 26 to 56 kDa. The most relevant connexins to cerebral artery myocyte function are Cx40, Cx43, and Cx45 (13, 30, 31). The partial cytoplasmic continuity provided by these aqueous intercellular channels are relatively nonselective and permeable to ions and second messenger molecules (up to approx 1,200 Da) (39, 44). Rafts of individual gap junction channels provide the structural correlate of intercellular communication, that is, the gap junction plaque. In this regard, gap junctions provide a low-resistance pathway through which intracellular signals can diffuse from activated cells to their coupled, unstimulated (or partially stimulated) neighboring cells. In this scenario, for example, alterations in intracellular second messenger levels produced by changes in the wall tension in a subpopulation of vascular smooth muscle cells may diffuse to and activate adjacent cells via gap junctions.

It has now become clear that intercellular communication through gap junctions is an important modulator of tone at all levels of the vascular tree (4, 7, 13). In resistance vessels, gap junctions are important for coordination of vasodilation and constriction and thus regional conducted changes (increases or decreases) in blood flow (37). In situ studies with arterioles demonstrate that local application of agonists such as acetylcholine or norepinephrine produces an upstream propagation of dilatory or constrictor responses. These responses could not be explained by diffusion of the agonists but could be inhibited by agents that interfere with gap junctional communication (38). In iridial arterioles, gap junctions are essential to coordination of spontaneous rhythmic contractions of myogenic origin (21). An important role for gap junctions in the spread of a hyperpolarization and vasodilation is reported in coronary arteries (2). Gap junctions may also be important in tonic contractile responses of rabbit mesenteric artery (10) and in rat aorta (13). Thus gap junction-mediated intercellular communication is an important modulator of myocyte tone in vessels throughout the vascular tree.

The goal of the present study was to investigate the role of intercellular communication in modulating the response of cerebral resistance arteries to distinct, but physiologically relevant, stimuli. To this end, we employed structurally distinct inhibitors of gap junction communication. Several "relatively selective" uncoupling agents have been characterized to date, but among these, heptanol (7, 10-12) and 18alpha -glycyrrhetinic acid (18alpha -GA) (14, 17, 42) are the most commonly used agents. Although peptide inhibitors of intercellular communication have more recently been employed, they have their apparent interpretational limitations as well (3). Therefore, we utilized heptanol and 18alpha -GA to evaluate the contribution of gap junctions to vasoconstrictor responses mediated by three distinct stimuli: 1) the myogenic response (secondary to changes in intraluminal pressure), 2) the receptor-mediated contractile response (agonist, e.g., vasopressin-induced depolarization and constriction), and 3) the depolarization-induced constrictor response (direct change in membrane potential by "voltage-clamp, see Ref. 24). In addition, we examined the combined effects of pressure and agonist [or depolarization with 80 mM K+-physiological salt solution (KPSS)]. The effects of heptanol and 18alpha -GA on the membrane potential in isolated, pressurized vessels were also investigated. In short, our data indicate that in intact resistance arteries, smooth muscle cells within the vascular wall may exhibit differential sensitivity to distinct contractile stimuli (i.e., heterogeneity of vascular smooth muscle responsiveness). The main physiological implications of the findings described in this study are that the syncytial vascular response to pressure- and agonist-induced contraction are dependent on intercellular communication through gap junctions. Moreover, inhibition of gap junctions reveals an inhomogeneity of membrane potential responses among individual myocytes within the vascular wall.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue Isolation and Preparation

Male Sprague-Dawley rats (300-450 g) were anesthetized with pentobarbital sodium (65 mg/kg ip) and administered heparin sulfate (500 IU ip). The brain was removed and placed into ice-cold physiological salt solution (PSS). Second-order middle cerebral arteries (150-250 µm) were dissected and transferred to a pressure arteriograph vessel chamber (Living Systems) containing oxygenated PSS maintained at room temperature. The superfusing solution was recirculated from an external bath at a flow rate of 15 ml/min. The measurements of pressure and diameter were performed as previously described (27). Briefly, one end of the vessel was mounted onto a cannula (tip diameter = 100 µm) and fixed by using a single nylon thread filament. The intraluminal pressure was minimally increased (3 mmHg) by using a pressure servo (Living Systems) to gently flush out residual blood; the opposite end was then similarly mounted and tied. Intraluminal pressure was elevated to 60 mmHg at which point the bath temperature was raised to 37°C after the absence of leaks was confirmed. The artery segment was equilibrated for 60 min at 60 mmHg during which time all vessels reliably developed pressure-induced myogenic tone. The vessel chamber was placed onto an inverted microscope, and vessel diameter was measured using a video edge-detection system (Living Systems). Intraluminal pressure and diameter were continuously acquired (DATAQ) and recorded onto a personal computer.

Experimental Protocols

Effect of heptanol on artery tone. To determine the functional role of gap junctions in myogenic tone, heptanol was added to the bath cumulatively (1.0 µM-3.0 mM). This range encompassed the previously reported selective concentration required for intercellular uncoupling (7). The tissue was exposed (2-3 min) to each concentration of heptanol, and the final maintained diameter was recorded. In some experiments, tissues were exposed to gap junction inhibitors (10-15 min) before the addition of vasoactive agents. Gap junction uncouplers used by either method (cumulative addition or preincubation) caused the same extent of inhibition. In a separate series of experiments, vasopressin (10 nM) or depolarizing 80 mM KPSS were alternately used as preconstricting agents to compare the inhibitory/uncoupling effects of heptanol on vessel tone. These experiments were conducted at both 20 and 60 mmHg, respectively. Reducing the transmural pressure to 20 mmHg places the vessel below the lower limit of the pressure range for myogenic reactivity. At the end of each experiment, 0 mM Ca2+ PSS was substituted to obtain the passive diameter of each vessel.

Effect of 18alpha -GA on artery tone. In some experiments, 18alpha -GA was used in place of heptanol. For these experiments, 18alpha -GA (1.0-50 µM) was added cumulatively to the recirculating bath in the presence of myogenic tone, agonist-, or depolarization-induced tone. This concentration range is reportedly selective for the inhibition of gap junctional communication (6, 17, 40-41). The effects of 18alpha -GA inhibition on KPSS- and agonist-induced constriction were examined at 20 mmHg. At the end of these experiments, the superfusate was replaced with 0 mM Ca2+ PSS to obtain the passive diameters.

Effects of gap junction inhibitors on membrane potential in pressurized arteries. To further examine the contribution of intercellular communication to myogenic tone, the effects of heptanol and 18alpha -GA were measured on the transmembrane potential of cerebral arteries by using the sharp electrode technique. Membrane potentials were recorded in intact pressurized arteries by using conventional intracellular glass microelectrodes filled with 3 M KCl solution and tip resistances of 40-60 MOmega as described previously (25). Cerebral arterial smooth muscle cells were impaled from the cleaned adventitial side of the pressurized artery. Membrane potentials were recorded with an Axoclamp 2B Amplifier (at 0.5-1 kHz bandwidth) at an acquisition rate of 1 kHz. Criteria for acceptance of recordings of successful impalements were 1) an abrupt change in potential on impalement of the cell, 2) stable membrane potential for at least 2 min, 3) unchanged tip resistance before and after impalement, and 4) tip potentials of <2 mV.

Drugs and Solutions

All drugs and materials were purchased from Sigma (St. Louis, MO). Heptanol and 18alpha -GA stock solutions were made fresh daily at stock concentrations of 1.0 M and 10 mM, respectively, and further diluted with the PSS to prepare the desired concentrations. 18alpha -GA was dissolved in DMSO. The concentration of the solvent in the PSS did not exceed 1/1,000. These procedures did not alter the pH of the solution or the pressure-diameter relation or the vascular responses to vasopressin or high KPSS. The composition of PSS was as follows (in mM): 118 NaCl, 4.7 KCl, 1.17 MgSO4, 1.18 KH2PO4, 24.9 NaHCO3, 1.6 CaCl2, 0.026 EDTA, and 11.1 glucose. Equimolar concentrations of KCl and NaCl were substituted to make KPSS. Zero millimolar Ca2+ PSS was identical to PSS with the exclusion of Ca2+ and the addition of 2.0 mM EGTA. All buffers were adjusted to pH 7.4 before each experiment and constantly aerated with 95% O2-5% CO2.

Expression of Results and Statistical Analysis

Vessel diameters were normalized and expressed either as percent inhibition or constriction using the following equation: percent constriction = 100% × (DCa-free - DPSS)/ DCa-free, where D is the arterial diameter in calcium-free solution or in PSS.

Vasodilator responses were expressed as percent increase in diameter from the initial diameter (due to myogenic tone) at the corresponding pressure: percent inhibition = 100% × (DCa-free - DA)/(DCa-free - D'A), where DA and D'A are active diameters in the absence and in the presence of inhibitor, respectively.

All data are expressed as means ± SE, and n represents the number of vessels. Fifty percent inhibitory concentration (IC50) values are expressed as means ± SE as determined from logarithmic values following iterative curve-fitting techniques with the convention four-parameter logistic equation by using SigmaPlot 2000 software. Comparisons between mean values were made using Student's unpaired t-tests. To determine whether responses to the inhibitors were different among myogenic-, agonist-, or depolarization-induced tone, group comparisons were made using two-way ANOVA and Fisher's protected least-significant difference test. A P value <0.05 was the basis for rejection of the null hypothesis. For the membrane potential studies, membrane potential values were determined as the average membrane potential measured over a 30-s, cursor-selected data segment. Data segments were taken before and after addition or removal of a substance when membrane potential had reached a new steady level. For this purpose, data files or ASCII files were imported into Origin (Microcal Software; Northampton, MA) by using the pCLAMP module of this program. Membrane potential values are expressed in millivolts as means ± SD samples of pooled N individual impalements from n different animals.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of Heptanol

Heptanol inhibited 80 mM KPSS-induced tone at 20 mmHg in a concentration-dependent manner (Fig. 1); the median inhibitory heptanol concentration (IC50) was 2.0 ± 0.7 mM (n = 4). The concentration-effect curve of heptanol for KPSS at 20 mmHg was steep (Hill slope = -2.6 ± 0.4) and is consistent with observations made by Rüdisüli and Weingart (36), who calculated a Hill slope of approximately equal to -2.3 for heptanol in cardiac myocytes. Vasopressin-induced constriction at 20 mmHg was significantly more sensitive to inhibition by heptanol than KPSS-induced constriction (IC50 = 400 ± 93 µM, n = 7, P < 0.007), and a significant inhibition was observed at concentrations as low as 1.0 µM (P < 0.05). Myogenic tone was also inhibited by heptanol (IC50 = 750 ± 90 µM, n = 8) and was more sensitive than KPSS-induced tone (P < 0.05). The concentration-response curve for heptanol inhibition of pressure-induced tone was not as shallow as that for inhibition of vasopressin responses (Hill slopes = -1.2 ± 0.1 and -0.4 ± 0.2, respectively). In fact, myogenic tone was inhibited at heptanol concentrations as low as 100 µM (P < 0.008) and was nearly completely abolished (97 ± 1%) at the highest tested concentration (3.0 mM).


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  		Fig. 1.   Heptanol inhibition of induced tone in the pressurized rat middle cerebral artery. A: representative trace of concentration-dependent heptanol inhibition of myogenic tone (at 60 mmHg). B: representative trace of concentration-dependent heptanol inhibition of 80 mM K+ physiological salt solution (KPSS)-induced vasoconstriction (at 20 mmHg). C: inhibitory log heptanol concentration-response relationships of myogenic (60 mmHg, n = 8), vasopressin-induced (20 mmHg, n = 7), and KPSS-induced (20 mmHg, n = 4) tones. Values are means ± SE. * P < 0.05 compared with KPSS. Myogenic tone at 60 mmHg is compared with the tones at 20 mmHg because myogenic tone at 20 mmHg is below the lower limit.

The effects of heptanol inhibition of vasopressin- and KPSS-induced tone at 60 mmHg were also examined (Fig. 2). At this intraluminal pressure, the arterial segments were myogenically active; vessels exposed to either of the preconstricting agents (vasopressin or KPSS) were additionally constricted by pressure. Therefore, the combination of these two distinct forms of vascular smooth muscle cell activation was used to further evaluate the sensitivity of these vessels to heptanol-induced disruption of intercellular communication. Heptanol inhibition of KPSS-induced constriction was unaffected by pressure and was not significantly different from the KPSS-inhibition curve at 20 mmHg (IC50 = 1.6 ± 0.5 mM, Hill slope = -1.99 ± 0.04, n = 7). However, the contractile response elicited by addition of vasopressin and myogenic tone was significantly less sensitive to heptanol inhibition than either form of stimulation alone. The IC50 for vasopressin-induced tone at 60 mmHg was 1.3 ± 0.4 mM (n = 4) and the corresponding Hill slope was -2.3 ± 0.4. 


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  		Fig. 2.   Heptanol inhibition of vasopressin and 80 mM KPSS in combination with myogenic tone at 60 mmHg. Inhibitory log heptanol concentration-response relationships of myogenic (60 mmHg, n = 8), vasopressin-induced (60 mmHg, n = 4), and KPSS-induced (60 mmHg, n = 7) tones. Values are means ± SE. * P < 0.05 compared with myogenic tone at 60 mmHg.

Effects of 18alpha -GA

The effects of 18alpha -GA on vascular tone were investigated by using concentrations from 1 to 50 µM. At concentrations greater than 50 µM, 18alpha -GA precipitated in the PSS. This insolubility prevented the determination of the maximum effect of 18alpha -GA. Therefore, IC50 for both vasopressin- and pressure-induced myogenic tones in the presence of 18alpha -GA could not be determined. The apparent IC50 values were thus estimated from the intercept of the curves with the 50% inhibition of effect. Eighty millimolar KPSS-induced tone was unaffected by 18alpha -GA (up to 50 µM) (n = 5; Fig. 3). In contrast, both vasopressin- and pressure-induced vasoconstrictions were significantly inhibited by 18alpha -GA at a threshold concentration of 3 µM (15 ± 4%, P < 0.04 and 5 ± 2%, P < 0.03; respectively). The effects of 18alpha -GA on vasopressin- and KPSS-induced tone at 60 mmHg was not examined for the following reasons. First, as mentioned earlier, 18alpha -GA (up to 50 µM) did not inhibit 80 mM KPSS-induced constriction at 20 mmHg. Thus tone due to the combination of KPSS and vasopressin at 60 mmHg would be higher (as seen in Fig. 2) than that to KPSS alone at 20 mmHg, which is unaffected by 18alpha -GA. Second, because 18alpha -GA precipitated in the PSS (at concentrations >50 µM), it would not be possible to investigate the effects of higher concentrations of 18alpha -GA on tone due to the combination of KPSS and vasopressin at 60 mmHg (as it was done in the case of heptanol).


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  		Fig. 3.   18alpha -Glycyrrhetinic acid (18alpha -GA) inhibition of induced tone in the pressurized rat middle cerebral artery. A: representative trace of vasoconstriction in response to a pressure step from 20 to 60 mmHg, followed by concentration-dependent inhibition of myogenic tone with 18alpha -GA. B: representative trace of 80 mM KPSS-induced vasoconstriction (at 20 mmHg) followed by increasing concentrations of 18alpha -GA. KPSS-induced tone was not inhibited by 18alpha -GA but was abolished when Ca2+ was removed from the extracellular solution. C: inhibitory log 18alpha -GA concentration-response relationships of myogenic (60 mmHg, n = 7), vasopressin-induced (20 mmHg, n = 4), and KPSS-induced (20 mmHg, n = 5) tones. Values are means ± SE. * P < 0.05 compared with KPSS.

Effects of Heptanol and 18alpha -Ga on Membrane Potential in Pressurized Cerebral Arteries

To further explore the role of intercellular communication in the modulation of resistance artery tone, we conducted a series of experiments designed to evaluate the effects of heptanol and 18alpha -GA on membrane potential in isolated, pressurized arterioles. The results are summarized in Table 1 and document that these compounds have little or no effect on membrane potential or arterial diameter at 20 mmHg (Fig. 4, trace 1). However, at 60 mmHg, there was a significant effect of both compounds on membrane potential (hyperpolarization) and a approx 50% vasodilation. Moreover, in contrast to the characteristic stability in membrane potential observed in these preparations in the absence of uncoupling agents, the presence of the uncoupling agents, in the same artery and in a few cases the same cell, we observed an apparently pressure-dependent increase in the number of impaled cells exhibiting transient, slow depolarizations of variable amplitude lasting several hundred milliseconds up to 1-2 s (see Table 1); that is, the number of cells exhibiting such oscillations increased from 5 of 28 impaled cells at 20 mmHg (i.e., 18%) to 9 of 23 impaled cells at 60 mmHg (i.e., 39%) (Table 2). Irrespective of transmural pressure, there was an apparent bimodal distribution of the slow wave depolarizations observed in the 14 cells studied. That is, 4 of 14 (i.e., 28%) cells exhibited slow waves with an amplitude and duration (means ± SD), respectively, of 7.6 ± 1.2 mV and 6.5 ± 0.6 s, whereas 10 of 14 (71%) cells had mean amplitude and duration values, respectively, of 1.3 ± 0.4 mV and 2.4 ± 0.8 s (Fig. 4, traces 2 and 3).

                              
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  		Table 1.   Effects of gap junction inhibitors on membrane potential of rat cerebral artery



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  		Fig. 4.   Effects of gap junction inhibitors on the membrane potential (Vm) of rat cerebral arteries. Most cells had stable Vm (trace 1, Table 1) in the presence of gap injunction inhibitors. However, in a subset of cells, the Vm was not stable and two different asynchronous oscillatory signals were observed (trace 2 and 3, Table 2).


                              
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  		Table 2.   Frequency of oscillations in membrane potential following cell impalement in the absence and in the presence of gap junction inhibitors


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Myogenic tone is a property that allows small arteries to autoregulate blood flow. This property has been demonstrated in a variety of animal and human vascular beds, including the cerebral, coronary, and renal systems. More specifically, increased intraluminal pressure causes an endothelium-independent depolarization of smooth muscle cells, the opening of L-type voltage-gated Ca2+ channels (24), and PKC activation (15, 28). The coordination of both myogenic- and agonist-induced constriction and dilation responses among vascular wall cells along the length of a blood vessel is critical to vascular homeostasis and function. However, the precise mechanism(s) responsible for such syncytial vascular smooth muscle responses is uncertain. This report documents a differential dependence of distinct agonist-induced contractions in isolated cerebral arteries on intercellular communication through gap junctions.

Evaluating Heptanol and 18alpha -GA as "Relatively Selective" Inhibitors

Heptanol and 18alpha -GA have frequently been used to investigate intercellular communication in intact smooth muscle preparations (1, 7, 9-11, 17, 31). Undeniably, these lipophilic agents lack specificity at high concentrations (i.e., concentration >1-3 mM). However, an algorithm for identifying reasonable experimental conditions under which such gap junction inhibitors may be utilized as a "relatively selective" uncoupling agents has been reported by us and others (1, 7, 9-11, 17, 31). Even under these conditions it is important to consider the possible spectrum of hypothesized nonjunctional activities of heptanol and 18alpha -GA. In this report, in which we studied steady-state contractile responses (i.e., tonic contractions), the most cogent possibilities for nongap-junctional effects include 1) changes in nonjunctional ion channel properties (e.g., altered Ca2+, K+, or Na+ current); 2) altered properties of direct or indirect second messengers; and 3) changes in myofilament calcium sensitivity and/or changes in the degree of myosin phosphorylation. Each of these possibilities is considered below.

Of particular importance is the fact that we are employing KCl (i.e., KPSS) as a benchmark for evaluating the gap junction independent portion of the contractile response in this preparation. That is, all myocytes respond similarly to depolarization, and there is no anticipated diffusion barrier in this preparation. We (24, 25) have shown previously that elevated external K+ acts as a "voltage clamp" in these small cerebral arteries due to the high levels of expression of inward rectifier K+ channels. In addition, the KPSS-induced contractile response of smooth muscle is accompanied by lower levels of myosin phosphorylation and tension development per unit increase in intracellular calcium level compared with an agonist-induced contraction (24, 35). The lack of effect of heptanol or 18alpha -GA to alter KPSS-induced contractile response is inconsistent with some possible effects that were unrelated to their activity on gap junctions. In this scenario, if heptanol or 18alpha -GA had a major action on smooth muscle contraction that was unrelated to its activity on gap junctions, then it stands to reason that application of heptanol or 18alpha -GA should universally alter the amplitude of the KPSS-induced contractile response. This was not the case in the present study (Figs. 1 and 3).

To further emphasize the point, we considered the fact that the average diameter of the vessels constricted with KPSS at 20 mmHg was equivalent to that observed with agonist- and pressure-induced constrictions at 60 mmHg (compare representative examples displayed in Fig. 1, A and B). The main physiological/pharmacological implication of this observation is that these equivalent contractile responses are nominally produced by equivalent stimuli. Given the presumed overlap in the distal second messenger/effector pathways presumably stimulated in this preparation by KPSS (translation of Ca2+ elevation into force production), agonist, or 60 mmHg, it is logical to conclude that the differential effect of heptanol on these contractile responses was due to the fact that the KPSS-induced responses were gap-junction independent, whereas the agonist and 60-mmHg responses were less so. A logical physiological correlate of this supposition is that significantly more myocytes respond to depolarization than to any given agonist or to pressure/stretch; i.e., the complement of membrane receptors/effectors is not the same on every cerebral myocyte. Viewed in this context, it is reasonable to conclude that the fact that heptanol and 18alpha -GA had no detectable effect on the magnitude of KPSS-induced contraction is inconsistent with the presence of a significant nonjunctional action at these concentrations.

Furthermore, in the present study, neither heptanol (1 mM) nor 18alpha -GA (20 µM) modified the membrane potential of smooth muscle cells in the rat middle cerebral artery at 20 mmHg. However, both compounds were associated with significant cellular hyperpolarization and corresponding increases in vessel diameter at 60 mmHg (Table 1). If one considers the likehood that 1 mM heptanol has no detectable effect on nonjunctional ionic currents in freshly isolated vascular myocytes (Christ and Wang, unpublished observations), then one cogent interpretation of our observations is that the decrease in membrane potential, as well as the increase in the number of cells exhibiting oscillations in membrane potential, could both result from a decreased level of intercellular communication. The rationale is as follows. If not every myocyte is capable of responding to changes in transmural pressure (or is temporarily refractory to the stimulus), then the responding cells generate cellular signals (i.e., voltage changes) that are propagated (conducted) through the intercellular pathway to adjacent cells. In turn, the amplitude of the change in membrane potential and the corresponding change in vessel diameter result from the averaged sum of the distributed responses of all of the responding pressure-sensitive cells contained in the functional vascular syncytium to all of the nonresponding cells. A precedent for such a modality in which individual cells, but not all cells, respond to a stimulus is set by the findings by Zang et al. (48) and Miriel and Knot (34). In this scenario, decreasing the extent of intercellular communication might be expected to decrease the "sphere of influence" of responding cells, thus decreasing the overall response of the vascular syncytium. The decreased signal spread would expect to be correlated with a more modest change in membrane potential (i.e., hyperpolarization) and a corresponding decrease in contractility (i.e., increased vessel diameter). At the same time, an increased resistance of the intercellular pathway might be expected to produce oscillations in membrane potential due to the decreased/increased restricted spread of the same stimulus. As such, if one accepts the presumption of the relatively selective uncoupling actions of heptanol and 18alpha -GA, then both the cellular hyperpolarization and the oscillations in membrane potential can be explained by decreased intercellular communication.

At the same concentrations of heptanol used in the present study, it was previously reported that heptanol did not affect the membrane potential in the guinea pig vas deferens (32). Our results reinforce those described above and suggest that under the conditions of our studies, both heptanol and 18alpha -GA are capable of selectively disrupting gap junction channels. In addition, electrophysiological and optical imaging techniques have shown that heptanol and 18alpha -GA are able to reversibly block junctional transfer of current-carrying ions, second messenger molecules, and fluorescent dyes in diverse cell types including vascular smooth muscle (1, 10, 14, 17, 40, 43, 46). Such cellular studies are encouraging, because they suggest the potential utility of using these lipophilic inhibitors, under appropriate experimental conditions, to probe the importance of intercellular communication to the regulation of tissue responses. A distinct strategy that uses peptide antibodies to evaluate the contribution of intercellular communication has also been investigated. There are both advantages and disadvantages to such an approach, and these have been recently reviewed elsewhere and, therefore, will not be further discussed herein (3).

Inhibition of Gap Junctions in Rat Cerebral Artery Wall

Again, if one accepts the premise that at lower concentrations (e.g., 100 µM-1 mM) both heptanol and 18alpha -GA are relatively selective and reversible gap junction uncoupling agents, then the observed heptanol-induced and 18alpha -GA-induced dilation of agonist and myogenic contractions can be interpreted to reflect disruption of recruitment of cells not directly activated by vasopressin or stretch. That is, the intercellular pathway is required to ensure syncytial smooth muscle responses.

The decreased ability of heptanol to inhibit vasopressin-induced contractile responses at 60 mmHg (Fig. 2) may reflect the synergistic effects of pressure and agonist stimulation on distinct and/or overlapping groups of cells. In this scenario, the vasoconstriction so obtained is less gap junction dependent because of the greater number of myocytes that are directly responsive to either vasopressin or stretch/pressure. Alternatively, such a phenomenon could reflect the additive effect of different second messengers produced by each stimuli (15) and/or potentiation of the effects of a nominally common second messenger pathway (26). More specifically, smooth muscle cells may not be uniformly sensitive to stimulation by any single modality, i.e., cells may respond to pressure or stretch but not both pressure-induced stretch and agonist (16). As such, junctional uncoupling may limit the transmission of electrical and/or chemical signals from directly activated cells to those cells that are more insensitive to a given stimulus, and therefore more dependent on intercellular signal transmission for activation. Viewed in this context, the combined effects of both agonist and stretch/pressure on distinct populations of cells would be expected to increase the effective number of directly activated cells.

Because cerebral arteries have an average diameter of 200 µm with up to four concentric smooth muscle layers across the media (29), it is quite possible that diffusion distances, tissue tortuosity factors and enzymatic degradation, and tissue uptake processes may converge to limit the effective diffusion radius of neuronal and endothelial derived substances in the vascular wall in vivo as well (7, 8, 10-12). Thus the presence of an intracellular mechanism to ensure a syncytial smooth muscle response makes sense. In fact, in the future it may be possible to estimate the fraction of responsive cells from the level of residual constriction provided by selective uncoupling concentrations of heptanol (see Fig. 4).

Perspectives

First, this study documents a differential effect of relatively selective gap junction uncoupling agents on the steady-state contractile responses of rat cerebral arteries elicited by KCl and receptor-mediated stimulation as well as a combination of myogenic- and receptor-mediated stimulation. These findings highlight the utility of heptanol and 18alpha -GA as pharmacological tools for investigating the role of gap junctions in vascular function and, furthermore, raise important issues concerning the potential physiological implications of heterogeneous responsivity of cerebral vascular wall cells to vasoactive agents (Fig. 5).


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  		Fig. 5.   Proposed scheme of heterogeneity in vascular wall cell sensitivity to activation by distinct receptor/effector mechanisms. A: all cells from the vessel wall responded to depolarizing solution (80 mM K+). B: fraction of cells from the arterial wall was able to respond to vasopressin; C: other cells were able to respond to change in intramural pressure. D: this proposed mechanism leads to heterogeneity of vascular smooth muscle cells.

Second, such results lend support to the notion that only a fraction of smooth muscle cells in the vessel wall need to be directly activated by any given modality of stimulation to ensure a syncytial vessel response; i.e., myocytes can be activated by agonists and/or by an increase of intraluminal pressure from the vessel lumen (34, 48). In this scenario, cells that are not directly responsive to any given modality would be recruited into the contractile response by junctional transfer of the receptor-mediated or pressure-mediated intracellular messengers/ions originating in directly responding smooth muscle cells within the vascular wall (see Fig. 4).

In summary, these data confirm and extend our previous studies and add to the growing body of experimental evidence consistent with the supposition that intercellular communication through gap junctions is an important modulator of the initiation, maintenance, and modulation of vascular tone and, as highlighted by the current report, is critical to the coordination of myogenic and agonist-induced constriction in cerebral resistance arteries (18, 21, 38).


    ACKNOWLEDGEMENTS

This work was supported by funds from the Heart and Stroke Foundation of Canada.


    FOOTNOTES

Address for reprint requests and other correspondence: G. J. Christ, Depts. of Urology & Physiology & Biophysics, Institute for Smooth Muscle Biology, Albert Einstein College of Medicine of Yeshiva Univ., Rm. 714, Forchheimer Bldg., 1300 Morris Park Ave., Bronx, NY 10461 (E-mail: christ{at}aecom.yu.edu).

This article belongs to a collection of papers accepted in response to the Editor's special call for papers entitled "Mechanisms of vascular myogenic tone."

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpheart.00605.2001

Received 10 July 2001; accepted in final form 15 July 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 283(6):H2177-H2186
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