INTRODUCTION

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ABDOMINAL ISCHEMIA reflexly excites the cardiovascular system predominantly by activating sympathetic A- and C-fiber afferents (18-20). Previously, we have shown that ischemic metabolites, including lactic acid, reactive oxygen species, prostaglandins (PG, e.g., PGE₂ and PGI₂), bradykinin, histamine, and serotonin, each contribute to the activation of abdominal visceral afferents during ischemia and reperfusion (8, 9, 20, 24, 29, 30). The precise mechanisms concerning their transduction processes, however, are unclear.

Activation of the phosphoinositide system through stimulation of receptors coupled to phospholipase C (PLC) by the guanine nucleotide-binding protein (G protein) is an important mechanism of intracellular signaling in sensory neurons throughout the body (2, 5). Activation of PLC cleaves the target membrane lipids to generate two second messengers: diacylglycerol and D-myo-inositol 1,4,5-trisphosphate. Diacylglycerol activates protein kinase C (PKC), whereas D-myo-inositol 1,4,5-trisphosphate mobilizes calcium from intracellular stores (21, 25). Together these processes initiate phosphorylation of cellular components, including membrane-bound receptors, ion channels, and enzymes (14, 23), all of which are key steps in the process of signal transduction (15).

At least three factors support a possible role of PKC in the regulation of neuronal activity. First, although PKC is distributed widely in various tissues and organs, it is most concentrated in the central and peripheral nervous system and is localized in both soma and terminals of neurons (5, 6, 14, 15). Second, PKC regulates a variety of neuronal cell functions (e.g., neuronal excitability, neurotransmitter release, and growth and differentiation) (6, 14, 22). Third, previous investigations suggest that PKC is involved in transduction of afferent signals. Specifically, data indicate that inhibition of PKC decreases hyperalgesia and C-fiber hyperexcitability (1). Moreover, perfusion of the renal pelvis with a phorbol ester that is known to be an activator of PKC [i.e., phorbol 12,13-dibutyrate; (PDBu)] increases activity of renal afferent nerves (16). Taken together, these studies indicate that PKC may contribute to activation of afferent nerves.

We have demonstrated that bradykinin produced during abdominal ischemia stimulates ischemically sensitive abdominal visceral afferents by activating kinin B₂ receptors (24). Bradykinin stimulates kinin B₂ receptors of sensory neurons, at least in part, by G protein-mediated activation of a phosphatidylinositol-specific PLC (2). As mentioned above, the intracellular second messenger diacylglycerol that subsequently activates PKC is generated from PLC (21, 25). Therefore, PKC may contribute to bradykinin-induced activation of abdominal visceral afferents. Results from several investigations suggest this possibility. For example, activation of PKC enhances bradykinin-induced stimulation of renal pelvic sensory receptors (16), and PKC inhibition reduces or abolishes bradykinin-induced neuronal activation (4). Whereas these results indicate that PKC may contribute to bradykinin-induced activation of afferent activity, direct evidence documenting a role for PKC in bradykinin-mediated stimulation of ischemically sensitive abdominal visceral afferents is lacking.

From our previous results (24) and those from other laboratories (1, 7, 16), the present study was designed to assess whether PKC contributes to the activation of ischemically sensitive abdominal visceral afferents during abdominal ischemia in the cat. Specifically, we tested the hypotheses that 1) exogenous activation of PKC stimulates ischemically sensitive abdominal visceral afferents; 2) inhibition of PKC attenuates the discharge activity of ischemically sensitive abdominal visceral afferents during abdominal ischemia; and 3) PKC contributes to bradykinin-mediated activation of ischemically sensitive abdominal visceral afferents. A preliminary report of this work has been published (11).

	METHODS

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Surgical Preparation

Our method for recording abdominal sympathetic afferent activity has been described in detail previously (24, 29, 30). Briefly, after we performed a midline sternotomy, the third through eleventh right ribs and the middle and lower lobes of the right lung were removed. After both phrenic nerves were cut, an inflatable occlusion cuff was placed around the descending thoracic aorta proximal to the diaphragm. This method to induce ischemia was used to eliminate the potential for collateral blood flow that may exist if a single artery was occluded (26). After we removed the overlying fascia, the right paravertebral sympathetic chain was isolated, draped over a Plexiglas platform, and covered with warm mineral oil. Next, small nerve filaments were dissected gently from the sympathetic chain between T₆ and T₁₀ using an operating microscope (Zeiss, Germany), and the caudal end was placed across a recording electrode. One pole of the recording electrode was grounded to the animal using a cotton thread. The recording electrode was attached to a high-impedance probe (model HIP511, Grass Instruments, Division of Astro-Med, Quincy, MA), and the signal was amplified (model P511 Preamplifier, Grass) and processed through an audioamplifier (AM8B, Audiomonitor, Grass) and an oscilloscope (model 2201, Tektronix, Beaverton, OR). Units were identified during the experiment using an oscilloscope to evaluate the size and configuration of the action potential in response to electrical stimulation of the receptive field. The neurogram and arterial pressure were recorded continuously (TA 4000B, Gould, Cleveland, OH). In addition, input from the neurogram was processed by a computer through an analog-to-digital interface card (R. C. Electronics, Santa Barbara, CA) to allow for subsequent off-line quantitative analysis. Discharge frequency of single-unit afferents was counted using custom-designed data acquisition and analysis software (version 3.02, EGAA, R. C. Electronics). If more than one afferent signal was obtained in response to any intervention, a window discriminator was used to identify the signal of interest. This function is incorporated into the EGAA program and was used during an off-line analysis.

Abdominal visceral organs were exposed through a midline abdominal incision. Receptive fields of afferent nerve fibers were located by placing a stimulating electrode directly onto the receptive field of each afferent to evoke electrically an action potential. The conduction time was determined by measuring the delay between the triggered artifact from electrical stimulation and the afferent's action potential detected by the recording electrode.

Conduction distance was estimated using a thread placed from the receptive field along the supposed afferent pathway through the paravertebral ganglion along the course of the major splanchnic nerve to the sympathetic chain to the recording electrode. The conduction velocity (CV) of each afferent was calculated by dividing the conduction distance by the conduction time. C fibers were classified as those with a CV < 2.5 m/s. Data presented in this paper were obtained from C fibers that had CV values ranging between 0.26 and 1.38 m/s. In addition, each C fiber had a receptive field that could be located precisely. After we completed the surgical instrumentation, the abdominal incision was closed and covered with warm saline-soaked gauze to minimize fluid and heat loss during the experimental protocols.

Experimental Protocols

In the same group of animals, afferent activity was measured in response to the appropriate volume-vehicle control for 2 µg/kg [i.e., 0.04% dimethyl sulfoxide (DMSO)], 5 µg/kg (i.e., 0.10% DMSO), and 8 µg/kg PDBu (i.e., 0.16% DMSO). The volume-vehicle control always was administered first because the effects of PDBu are potent and long lasting (20-70 min; 16, 28).

In a separate group of cats, afferent activity of four ischemically sensitive C fibers was assessed during a 10-min control period and after injecting phorbol-12,13-didecanoate (PDD; 8 µg/kg) through the femoral artery catheter into the descending thoracic aorta. PDD, an inactive yet structurally similar phorbol ester (16), was administered as an additional control to verify the effects of PDBu. If the activity of afferents was unaltered by PDD, PDBu (8 µg/kg ia) was injected 10-15 min later to verify the responsiveness of the ischemically sensitive C fiber.

Effect of PKC inhibition on afferent activity during ischemia. After we identified a C fiber, the discharge activity was measured during 10 min of abdominal ischemia followed by 5 min of reperfusion. If the afferent was ischemically sensitive, a second bout of ischemia was repeated 30-40 min later in the presence of selective PKC inhibition using PKC-(1936) (20 µg/kg iv, n = 7). In cases when the afferent activity was suppressed completely, the receptive field was manipulated mechanically or stimulated electrically to document viability of the nerve ending.

To demonstrate reproducibility, activity of C fibers was recorded in response to two bouts of ischemia that were separated by 30-40 min (n = 5). The vehicle for PKC-(1936) (i.e., 0.9% saline) was administered before the second exposure to ischemia.

Effect of inhibition of PKC on bradykinin-mediated activation of ischemically sensitive abdominal visceral afferents. After identifying an ischemically sensitive C fiber, we measured the response to bradykinin (0.5-0.1 µg/kg ia) in the absence and presence (20-30 min later) of selective PKC inhibition using PKC-(1936) (20 µg/kg iv, n = 13). The viability of each nerve was verified as described above.

To demonstrate reproducibility, the response of the afferent to two injections of bradykinin (0.5-1.0 µg/kg ia), separated by 20-30 min, was examined in seven animals. We previously showed that tachyphylaxis to repeated administration of bradykinin does not occur if injections are separated by 20-30 min (24). The vehicle for PKC-(1936) (i.e., 0.9% saline) was administered before the second exposure to bradykinin.

Drugs and Solutions

Data Analysis

Statistical Analysis

	RESULTS

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Effect of Exogenous PKC Activation on Activity of Ischemically Sensitive Afferents

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Responses of ischemically sensitive C fibers to phorbol 12,13-dibutyrate (PDBu). Histograms represent means ± SE from 23 fibers (2 µg/kg, n = 8; 5 µg/kg, n = 11; 8 µg/kg, n = 4). * P < 0.05 vs. respective control value.  P < 0.05, 8 vs. 2 µg/kg of PDBu.

The inactive phorbol ester PDD (8 µg/kg ia) did not increase afferent activity in any of the ischemically sensitive C fibers (n = 4, CV = 0.57 ± 0.06 m/s). All of these fibers, however, were stimulated subsequently by PDBu (8 µg/kg ia).

An original tracing of an ischemically sensitive C fiber (CV = 0.82 m/s) that responded to PDBu is shown in Fig. 2. Afferents studied in these protocols were located in the duodenum, gallbladder, mesentery, pancreas, and porta hepatis (Table 1).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Responses of an ischemically sensitive C fiber [conduction velocity (CV) = 0.82 m/s] innervating the pancreas to vehicle (0.10% dimethyl sulfoxide, DMSO; A) and PDBu (5 µg/kg; B). Neurograms 1-4 represent original tracings obtained at points 1-4 shown on A (1, 2) and B (3, 4).

Effect of Selective Inhibition of PKC on Ischemically Sensitive C Fibers

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Responses of ischemically sensitive C fibers to 2 bouts of ischemia (n = 5; A) and ischemia in presence of protein kinase C (PKC) inhibition using PKC-(1936) (20 µg/kg iv, n = 7; B). Histograms represent means ± SE. * P < 0.05 vs. respective control value.  P < 0.05 vs. first ischemia.

In five C fibers (CV = 0.47 ± 0.05 m/s), we examined the response to repeated ischemia without the PKC inhibitor. Comparing the first and second bouts of ischemia, we observed similar increases in afferent activity from baseline (0.07 ± 0.02 to 0.57 ± 0.11 impulses/s and 0.06 ± 0.03 to 0.57 ± 0.08 impulses/s; Fig. 3A), comparable decreases in femoral arterial pressure (89 ± 10 to 16 ± 1 mmHg and 89 ± 12 to 16 ± 2 mmHg), and onset latencies that were not different (179 ± 45 and 113 ± 32 s), respectively. An original tracing of an ischemically sensitive C fiber tested before and after inhibition of PKC (CV = 1.0 m/s) is shown in Fig. 4. Afferents studied in this protocol were located in the duodenum, mesentery, pancreas, and porta hepatis (Table 1).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Responses of an ischemically sensitive C fiber (CV = 1.0 m/s) innervating mesentery to 10 min of ischemia before (A) and after (B) inhibition of PKC using PKC-(1936) (20 µg/kg iv). Neurograms 1-4 represent original tracings obtained at points 1-4 shown on A (1, 2) and B (3, 4).

Effect of Selective Inhibition of PKC on Bradykinin-Mediated Discharge Activity of Ischemically Sensitive Afferents

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Responses of ischemically sensitive C fibers to repeated exposures to bradykinin (0.5-1.0 µg/kg ia, n = 7; A) and bradykinin following inhibition of PKC with PKC-(1936) (20 µg/kg iv, n = 13, B). Histograms represent means ± SE. * P < 0.05 vs. respective control value.  P < 0.05 vs. first injection of bradykinin.

The response to repeated administration of bradykinin (0.5-1.0 µg/kg ia) demonstrated similar increases from baseline in afferent activity of ischemically sensitive C fibers comparing the first (0.04 ± 0.02 to 1.00 ± 0.23 impulses/s) and second (0.08 ± 0.04 to 1.03 ± 0.22 impulses/s) injections (n = 7, Fig. 5A). Likewise, the onset latencies were not different comparing the first (15 ± 4 s) and second (16 ± 4 s) injections of bradykinin. An original tracing of an ischemically sensitive C fiber (CV = 0.37 m/s) is shown in Fig. 6. Afferents studied in this protocol were located in the duodenum, gallbladder, mesentery, pancreas, porta hepatis, and stomach (Table 1).

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Responses of an ischemically sensitive C fiber (CV = 0.37 m/s) innervating the pancreas to bradykinin (0.5 µg/kg ia) before (A) and after (B) inhibition of PKC using PKC-(1936) (20 µg/kg iv). Neurograms 1-4 represent original tracings obtained at points 1-4 shown on A (1, 2) and B (3, 4).

	DISCUSSION

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Results from the present study support our hypothesis that PKC contributes to activation of ischemically sensitive abdominal visceral C-fiber afferents. In this regard, we observed that exogenous activation of PKC increases the discharge activity of ischemically sensitive fibers, whereas PKC inhibition attenuates the responsiveness of these afferent fibers to abdominal ischemia. Our findings suggest that PKC is relevant physiologically, because bradykinin-mediated activation of abdominal visceral C fibers was suppressed by inhibition of PKC. Taken together, these results provide new information that PKC contributes to the signal transduction mechanisms of ischemically sensitive abdominal visceral afferents.

PKC is present mainly in the cytosol and is an important mechanism of signal transduction (15, 25). In general, when cells are stimulated, the concentration of diacylglycerol rises transiently, and PKC is translocated from the cytosol to the cell membrane where it is activated (15). Once activated, PKC phosphorylates cellular proteins, including cell surface receptors, cytoskeletal proteins, ion channels, and cytosolic and nuclear proteins (14, 23). By phosphorylating these proteins, PKC causes cell signaling both locally, at the site of activation, and globally by amplifying other signaling pathways such as cAMP, cGMP, calcium, and arachidonic acid metabolites (21). PKC also may be involved in the control of ion movement (e.g., Ca²⁺, Na⁺, and K⁺), thereby modulating the concentration of intracellular ions, membrane potential, and electrical signals (21). Moreover, additional reports indicate that PKC regulates a variety of neuronal cell functions such as neuronal excitability, neurotransmitter release, growth, and differentiation (6, 10, 14, 22, 23). Taken together, results from the se studies provide a substantial rationale for hypothesizing that PKC contributes to activation of ischemically sensitive abdominal visceral afferents.

To evaluate the potential for PKC to contribute to the transduction process(es) of ischemically sensitive C fibers, we determined first whether exogenous stimulation of PKC increases the afferent discharge of these particular fibers. PDBu was chosen for this purpose, because it is similar in molecular structure and physiological function to diacylglycerol (5, 15, 17, 23) and has been used previously to activate PKC (25). Previous data from other laboratories demonstrate that PDBu augments the activity of slowly conducting single-unit afferents of the knee joint of the cat when it is administered intra-arterially (28) and whole nerve activity of renal afferent nerves when it is applied to the renal pelvis in the rat (16). Our results indicate that PDBu also is capable of increasing the discharge rate of ischemically sensitive abdominal visceral C fibers in a dose-dependent manner. For example, afferent activity increased 5-, 17-, and 22-fold in response to 2, 5, and 8 µg/kg of PDBu, respectively. These data, together with those indicating the inability of time, vehicle, and/or the inactive phorbol ester (PDD) to alter afferent activity, confirm previous reports that PDBu can penetrate cells to stimulate PKC, which, in turn, increases afferent nerve activity (16, 28). In addition, our results provide new and important findings that exogenous activation of PKC specifically is capable of stimulating a subgroup of abdominal C-fiber afferents that are ischemically sensitive.

Next, we assessed whether activation of endogenous PKC during abdominal ischemia contributes to stimulation of C fibers. To evaluate the importance of PKC, we recorded afferent nerve activity during abdominal ischemia in the absence and presence of PKC inhibition using PKC-(1936). PKC-(1936) is a pseudosubstrate peptide inhibitor that binds to the active sites of PKC in a potent and selective manner (1, 3, 13, 27). Intradermal injection of PKC-(1936) has been shown to decrease hyperalgesia and C-fiber hyperexcitability in the streptozotocin-diabetic rat (1). Consistent with our hypothesis, we observed that ischemia-induced increases in afferent activity were attenuated in the presence compared with the absence of PKC inhibition. The control studies further strengthen these findings, since increases in discharge activity were similar in response to two bouts of abdominal ischemia. Therefore, the attenuated response to ischemia during PKC inhibition cannot be attributed to reduced fiber responsiveness, tachyphylaxis to ischemia, and/or a time-vehicle effect. These are the first data to demonstrate that PKC activation contributes to stimulation of visceral afferents during abdominal ischemia.

Previously, we demonstrated that mesenteric ischemia leads to the production of several substances, including bradykinin, reactive oxygen species, lactic acid, cyclooxygenase products, serotonin, and histamine, that contribute to the activation and/or sensitization of ischemically sensitive abdominal visceral afferent nerve endings (8, 9, 20, 24, 29, 30). The precise mechanism(s) concerning the transduction process(es) of these products, however, is unclear. In the present study, we sought to determine the importance of PKC in the activation of visceral afferent C fibers in response to bradykinin. Bradykinin was chosen because it 1) increases during abdominal ischemia (24); 2) stimulates ischemically sensitive abdominal visceral afferents (24); and 3) potentially activates neurons through a PKC-mediated mechanism (2, 7). We observed that administration of bradykinin into the descending thoracic aorta increased the discharge frequency of ischemically sensitive C fibers from baseline by a similar extent as reported previously (24). However, after inhibition of PKC, the bradykinin-related increase in impulse activity from baseline was attenuated by 44%. Similar to our previous protocol using PKC-(1936), control experiments verified that the smaller response to bradykinin observed after PKC inhibition was not due to reduced fiber responsiveness, tachyphylaxis to bradykinin, and/or a time-vehicle effect. Taken together, these data support our hypothesis that PKC contributes to bradykinin-mediated activation of ischemically sensitive abdominal visceral afferents.

Although the response of visceral afferents to both abdominal ischemia and bradykinin was attenuated after PKC inhibition, it was not abolished. This suggests that whereas PKC makes an important contribution to activation of ischemically sensitive C fibers, it likely is not solely responsible for their stimulation. Other intracellular messengers such as cAMP, cGMP, and calcium-mobilizing agents (e.g., D-myo-inositol 1,4,5-trisphosphate) are capable of phosphorylating cellular proteins through protein kinase A, protein kinase G, and Ca²⁺-calmodulin kinases, respectively (12, 25). Because of the inherent redundancy that exists concerning intracellular messaging, it is not surprising that PKC inhibition alone was unable to abolish completely the response of abdominal visceral C fibers to ischemia or bradykinin. However, our data provide strong evidence that activation of PKC contributes significantly to bradykinin-related activation of ischemically sensitive abdominal visceral afferents.

Concern may be raised that the onset latency of afferent activation was longer after PDBu (205 s) compared with ischemia (185 s) and bradykinin (14 s). The increased duration required for exogenous stimulation of C fibers may reflect the time required for the phorbol ester to reach the sensory endings, travel intracellularly, and activate PKC. Conversely, during exogenous administration and ischemia-induced production of bradykinin, afferents are activated directly through kinin B₂ receptors. Therefore, the different mechanisms of PKC activation (exogenous vs. endogenous) likely explain the dissimilar times required for afferent activation.

Abdominal ischemia causes profound cardiovascular reflexes characterized by increased blood pressure, heart rate, and myocardial contractility. For instance, blood pressure typically increases by 35-55 mmHg during abdominal ischemia (26). The physiological importance of this reflex likely functions to increase the chronotropic and inotropic properties of the heart to augment cardiac output and maintain blood pressure, thus facilitating perfusion to ischemic visceral organs. Abdominal visceral C fibers form the afferent limb of this reflex and are stimulated by a variety of substances produced during ischemia. Whereas many of these substances are known, the precise mechanisms regarding their transduction processes and ultimate activation of visceral C fibers are unclear. Results from the present study provide important new information indicating that PKC contributes to activation of ischemically sensitive abdominal visceral C fibers. Exogenous activation of PKC using PDBu stimulates, whereas inhibition of PKC using PKC-(1936) attenuates, the discharge rate of ischemically sensitive abdominal visceral afferents. In addition, our findings indicate that bradykinin-mediated activation of ischemically sensitive afferents is attenuated by inhibiting PKC activity. These data indicate that PKC contributes to the activation of abdominal visceral afferents during ischemia and specifically to bradykinin-related activation of these afferents.

In perspective, several metabolites produced during abdominal ischemia (e.g., lactic acid, bradykinin, histamine, and serotonin) contribute to activation of sensory nerve endings that form the afferent limb of the reflex pressor response. The precise mechanisms by which these metabolic substances act and the methods of signal transduction utilized, however, are unclear. The results of the present study indicate that PKC contributes to the activation of ischemically sensitive abdominal afferents. Specifically, PKC plays an important role in bradykinin-mediated activation of these afferents. These findings suggest strongly that the phosphoinositide system is one of the intracellular signaling pathways used when abdominal visceral sensory receptors are stimulated by bradykinin. These data contribute to our understanding of the chemical-signaling mechanisms underlying activation of abdominal visceral afferent nerve endings in response to ischemia.