Intrathecal injection of cell-permeable analogs of cyclic 3',5'-guanosine monophosphate produces hyperalgesia in mice.

Several recent studies suggest that spinal cord levels of cyclic 3',5'-guanosine monophosphate (cGMP) may participate in the development of hyperalgesia. The purpose of this study was to directly evaluate whether cell permeable analogues of cGMP evoke a thermal hyperalgesia (using a hot-plate assay) when administered intrathecally in mice. Our results indicate that two cell permeable forms of cGMP evoke a dose dependent hyperalgesia when administered intrathecally in mice. Additionally, this hyperalgesia was selective since neither non-cell permeant cGMP nor guanosine had any effect on the latency of paw withdrawal when compared to the vehicle injected controls. These data indicate that cGMP is involved in the facilitation of thermal hyperalgesia at the level of the spinal cord.

Autoradiographic analysis of 125I-substance P binding in rat spinal cord following chronic constriction injury of the sciatic nerve.

Using receptor binding and autoradiographic techniques, changes in Bolton-Hunter labeled 125I-substance P (125I-BH-SP) binding were determined in laminae I/II, V and X of rat lumbar spinal cord after chronic constriction injury (CCI) of the sciatic nerve. When compared to the sham-operated side of the control group, SP binding significantly increased ipsilateral to the CCI in laminae I/II at 5, 10 and 20 days after injury and in lamina V at 5 days after injury. Scatchard analysis was performed on the 125I-BH-SP binding to the NK1 receptor in laminae I/II of rats 5 days after generation of the CCI. A significant decrease in the Kd of 125I-BH-SP binding was observed in laminae I/II ipsilateral to CCI when compared with the control side (ipsilateral to sham surgery). There was no significant change in the Bmax in laminae I/II ipsilateral to CCI. The changes in 125I-BH-SP binding in the rat spinal cord that occurred after CCI were found in areas of the spinal cord that receive terminations of nociceptive primary afferent fibers. The increased affinity of the NK1 binding site that we report could result in an increase in SP receptor activation in laminae I/II. Such central changes in SP binding may contribute to the neuropathic pain syndrome observed in rats with the CCI.

Characterization and autoradiographic localization of gastrin releasing peptide receptors in the porcine gut.

The biochemical characteristics of specific binding sites for gastrin releasing peptide were examined in a preparation of the mucosa and submucosa of the porcine jejunum. Iodinated gastrin releasing peptide bound to sites in this preparation with an affinity that was similar to its potency in stimulating anion transport in the epithelium. The relative order of potency of peptide analogues of gastrin releasing peptide in contracting longitudinal smooth muscle was similar to their order of potency in altering anion transport across the mucosa. Autoradiographic studies showed that specific binding sites for [125I]gastrin releasing peptide were associated with the epithelium of the mucosa as well as the myenteric plexus. Gastrin releasing peptide-like immunoreactive axons paralleled the distribution of specific binding sites for the peptide. These data support a physiological role for gastrin releasing peptide in the control of intestinal ion secretion and motor function.

Neurotensin binding sites in porcine jejunum: biochemical characterization and intramural localization.

Neurotensin is present in high concentrations in the mammalian gut, especially in enteroendocrine cells of the mucosa. Exogenous neurotensin has been shown to alter ion transport by the mucosa and contractile activity of intestinal smooth muscle. The purpose of this study was to determine the distribution of neurotensin binding sites within the intestinal wall. Initially, biochemical characteristics of [125I]neurotensin binding sites were determined within two preparations of the distal porcine jejunum: (1) the mucosa and submucosa, and (2) the circular and longitudinal muscle with their intramural plexuses. Ligand binding data for the preparation including the mucosa and submucosa indicated that [125I]neurotensin bound specifically to two sites having apparent equilibrium dissociation constants of approximately 0.046 and 0.37 nM. A binding site with a dissociation constant of approximately 0.38 nM was confirmed for the preparation of muscle and associated intramural plexuses. Xenopsin and neurotensin were equipotent to neurotensin in competing for these binding sites; neuromedin N was approximately 40 times less potent in the preparation of mucosa and submucosa. Receptor autoradiography was used to determine the distribution of [125I]neurotensin binding sites within the wall of the jejunum. Autoradiograms of [125I]neurotensin bound to cross sections of the proximal and distal jejunum showed that the highest densities of silver grains were associated with the internal submucosal ganglia, external submucosal plexus and myenteric ganglia. A moderate density of silver grains was associated with the circular muscle. The localization of neurotensin binding sites to submucosal ganglia is consistent with observations that neurotensin effects on active anion secretion by the mucosa are blocked by tetrodotoxin. Immunohistochemical localization of neurotensin in the porcine jejunum demonstrated a limited population of neurotensin immunoreactive cells within the mucosal epithelium. It is possible that neurotensin released from these cells in the mucosa as well as neurotensin-related peptides released from enteric neurons may be the endogenous ligands for the binding sites visualized in this study.

Muscimol, gamma-aminobutyric acidA receptors and excitatory amino acids in the mouse spinal cord.

These experiments examined the effects of intrathecally administered gamma-aminobutyric acid (GABA) agonists on the effects of intrathecally administered excitatory amino acid (EAA) agonists: N-methyl-D-aspartic acid (NMDA), quisqualic acid and kainic acid. We have found that muscimol, a GABAA receptor agonist, but not baclofen, a GABAB receptor agonist, dose-dependently inhibited caudally directed biting and scratching behavior induced by all three EAA agonists. This nonselective blockade of the expression of effects mediated by all three types of EAA receptor is in marked contrast to the selective blockade of NMDA effects seen previously in the case of mu opioids and phencyclidine receptor agonists. Inhibition by muscimol was blocked with the GABAA receptor antagonist, bicuculline. Decreased latency or hyperalgesia in the tail-flick test, found previously to be induced selectively by NMDA and blocked by an NMDA receptor antagonist, was similarly affected by muscimol but not baclofen, each given intrathecally. However, muscimol prolonged the tail-flick latency only after presentation of NMDA suggesting a possible antinociceptive effect of GABAA agonists in the presence of agonists at NMDA receptors. This study together with the preceding paper resolves GABA-mediated spinal antinociception into two components: a GABAA agonist selectively blocks nociception involving EAA receptors whereas a GABAB agonist selectively blocks substance P spinal activity (the preceding paper).

Phencyclidine and sigma receptors in rat spinal cord: binding characterization and quantitative autoradiography.

These experiments were designed to compare phencyclidine (PCP) and sigma (sigma) receptor binding sites in the rat spinal cord by using receptor binding and autoradiographic techniques. Binding sites for 3H-TCP (3H-1-[1-(2-thienyl)cyclohexy]piperidine), a PCP receptor agonist, and (+)3H-3-PPP (3H-(+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine), a sigma receptor agonist, in the rat spinal cord were shown to represent two populations of recognition sites. Inhibition studies revealed that ligands with high affinity for the PCP receptor (MK-801 and PCP) were potent competitors at 3H-TCP binding sites whereas the putative sigma receptor ligands (+/-)pentazocine and haloperidol were potent competitors at (+)3H-3-PPP binding sites. The autoradiographic distribution of 3H-TCP and (+)3H-3-PPP binding sites in adjacent sections of rat spinal cord demonstrated the occurrence of two distinct populations of binding sites. 3H-TCP binding sites were localized primarily in laminae I and II in cervical and thoracic spinal segments. Binding sites in lamina I decreased in density along a rostral to caudal gradient in the spinal cord. The highest density of (+)3H-3-PPP binding sites was found in the ventral horn (lamina VIII and IX) and over perikarya in dorsal root ganglia. Significantly elevated densities of (+)3H-3-PPP binding sites were also found in lamina X within thoracic and lumbar segments and in the intermediolateral cell column. The results of the present study show that PCP and sigma receptor binding sites are differentially localized in the rat spinal cord and suggest that separate binding sites exist for PCP and sigma agonists.

Nociceptive action of excitatory amino acids in the mouse: effects of spinally administered opioids, phencyclidine and sigma agonists.

Intrathecal administration of the excitatory amino acid (EAA) agonists, N-methyl-D-aspartate (NMDA), quisqualate (Quis) or kainic acid (KA), in the spinal subarachnoid space of mice produced a dose-related biting and scratching behavior. Higher doses appeared aversive, suggesting a nociceptive action for EAAs in the spinal cord. Intrathecally administered NMDA, but not Quis or KA, produced a hyperalgesic effect in the tail-flick and hot-plate tests. To test the hypothesis that EAA agonists are involved in transmission of nociceptive information in the spinal cord, we tested the effect of various opioid, sigma and phencyclidine compounds on the action of NMDA in the tail-flick, hot-plate and biting and scratching nociceptive tests. Our results indicated that the involvement of mu, sigma and phencyclidine receptors was predominant in blockade of the behavioral and hyperalgesic effects of intrathecally administered NMDA. Delta receptors appeared less involved, and involvement of kappa receptors was not detectable in blockade of the behavioral and hyperalgesic effects of intrathecally administered NMDA. Quis and KA effects were not altered by any of these agonists. Agonist doses required to inhibit NMDA-induced hyperalgesia in the tail-flick and hot-plate tests were significantly less than those needed to inhibit biting and scratching behavior. The adrenergic agonist norepinephrine inhibited NMDA- but not Quis- or KA-induced biting and scratching behavior. This action appeared to be alpha-1 mediated because it was reversed by phentolamine but not by yohimbine. These results suggest that the actions of EAAs in the spinal cord are differentially affected by various opioid phencyclidine, sigma and adrenergic receptor agonists and support the hypothesis that EAAs are involved in the transmission of nociceptive information in the spinal cord.

Phencyclidine selectively blocks a spinal action of N-methyl-D-aspartate in mice.

Excitatory amino acids (EAAs) administered intrathecally (i.t.) in the mouse elicit a caudally directed biting and scratching behavior. N-methyl-D-aspartate (NMDA) is a potent agonist that produces this behavior, and its action is inhibited by the NMDA receptor antagonist, D-2-aminophosphonovaleric acid (D-APV). The behavioral response to the agonists resembles the response to i.t. substance P (SP) except that the response to the EAAs is more intense and, at higher doses, is accompanied by vocalization. The behavioral response to i.t. EAAs is not enhanced by i.t. SP. Whereas [D-Ala2-D-Leu5]enkephalin (DADL) and norepinephrine (NE) inhibit SP-induced biting and scratching, these compounds only partially inhibit EAA-induced behavior. Phencyclidine (PCP), on the other hand, inhibits completely NMDA-induced behavior but not behavior induced by SP or other EAAs. We conclude that the behavior induced by i.t. EAAs is mediated by spinal EAA receptors and that this response is pharmacologically distinct from responses to SP.

Pharmacokinetics of verapamil: experience with a sustained intravenous infusion regimen.

Disappearance kinetic characteristics of verapamil were determined in 9 patients after a single intravenous dose. From the pharmacokinetic variables determined, we designed an intravenous regimen to maintain a plasma verapamil concentration of 150 ng/ml consisting of (1) a loading bolus (10 mg over 2 minutes), followed by (2) a rapid loading infusion (0.375 mg/min) for 30 minutes, and finally (3) a maintenance infusion (0.125 mg/min). We tested this regimen in 7 patients for 2 to 12 hours, and found it to be safe and to produce stable prolongation of the P-R interval. Verapamil concentration was highest immediately after the bolus administration and was prevented from falling below 67 ng/ml by the rapid infusion. Maintenance concentration remained between 77 and 156 ng/ml for all patients, and averaged 122 ng/ml. Transient and slight decreases in brachial blood pressure and sinus cycle length occurred coincident with the maximum verapamil concentration. Maximum P-R prolongation lagged behind peak plasma concentration but was sustained for the duration of the infusion. Prolongation of the P-R interval was not significantly different at the end of the infusion from that 90 minutes after the start of the regimen. No patient demonstrated significant side effects, arrhythmia, or clinically important hypotension. Although the specified regimen produced a final concentration averaging 125 ng/ml, it is predicted that infusion regimens producing other plasma concentrations can be similarly devised by changing the bolus, rapid loading infusion, and maintenance infusion doses in proportion to the desired final plasma concentration.