omega-Conotoxin exerts functionally distinct low and high affinity effects in the neuronal cell line NG108-15.

The Ca2+ channel blockade produced by omega-conotoxin GVIA (omega-CgTx) was studied in single, forskolin-differentiated, NG108-15 cells, using dual-emission microfluorimetry and the whole-cell patch-clamp technique. Whole-cell currents through Ca2+ channels were measured with 5 mM Ba2+ as the charge carrier. Application of 1 microM nitrendipine inhibited by 90% the currents evoked by stepping from -30 mV to 0 mV. Omega-CgTx (1 microM) inhibited these currents by 28%. These data suggest the possibility that NG108-15 cells express two types of dihydropyridine-sensitive Ca2+ channel, one sensitive and the other insensitive to blockade by omega-CgTx. The nature of the Ca2+ channel blockade produced by these agents was studied further, using depolarization-induced intracellular free Ca2+ concentration [( Ca2+]i) transients recorded with the Ca2+ indicator indo-1 and a dual-emission microfluorimeter. A 30-sec superfusion with 50 mM K+ increased the [Ca2+]i from a basal level of 142 +/- 10 nM to a peak level of 1655 +/- 287 nM. This [Ca2+]i transient was blocked completely and reversibly by nitrendipine, in a concentration-dependent manner (IC50 = 1.9 nM). In contrast, omega-CgTx produced a maximal inhibition of the depolarization-induced rise in [Ca2+]i of only 52% in the presence of physiological concentrations of divalent metals. The block was irreversible. This inhibition was concentration dependent until the point of maximal inhibition, at which point the channel block reversed in a graded manner. This entire U-shaped dose-response curve could be shifted in a parallel fashion by modulation of the extracellular divalent metal concentration, without changes in the maximal inhibition. Repeated applications of or prolonged incubations with omega-CgTx failed to increase the maximal block. Treatment with a high (1 microM) concentration of omega-CgTx, which produced a modest (10%) inhibition of Ca2+ influx, protected the cell from a second exposure to a normally effective concentration of omega-CgTx (10 nM). Depolarization-induced [Ca2+]i transients in cells treated with 10 nM toxin were inhibited by 45%, and this inhibition could not be reversed by subsequent exposure to a high concentration of omega-CgTx. We conclude that there are two omega-CgTx binding sites on these cells, one to which omega-CgTx binds with high affinity, producing an irreversible Ca2+ channel blockade, and a second to which omega-CgTx binds with lower affinity. Binding to this second site is irreversible and does not block the channel but does prevent access to the high affinity site.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of calcium currents in cultured myenteric neurons by neuropeptide Y: evidence for direct receptor/channel coupling.

Single channel recordings from rat myenteric plexus neurons demonstrated the presence of two categories of Ca2+ channels. One type of Ca channel had a slope conductance of 27 pS and was sensitive to dihydropyridines while the other channel type had a conductance of 14 pS and was dihydropyridine-insensitive. The 14 pS channel was mostly inactivated at a holding potential of -40 mV, while the 27 pS channel was much more resistant to depolarized holding potentials. A majority of whole-cell current was reprimed by the use of negative holding (-90 mV) potentials, when compared to that obtained at a holding potential of -40 mV. These properties are consistent with N- and L-type Ca channels previously described. In general, the inactivating part of the whole-cell Ca2+ current, selectively reprimed by negative holding potentials, was inhibited by neuropeptide Y (NPY). Depolarization-induced [Ca2+]i transients assessed using fura-2 showed that the inhibitory effects of nitrendipine and NPY were additive. The effects of NPY were abolished by pertussis toxin pretreatment. Single-channel experiments showed that neither the 14 nor the 27 pS Ca channel currents were inhibited by the addition of NPY outside the patch pipette. These results suggest that NPY modulates N-type Ca2+ channels selectively in these neurons and that an easily diffusible second messenger does not appear to participate in receptor/channel coupling.

The role of caffeine-sensitive calcium stores in the regulation of the intracellular free calcium concentration in rat sympathetic neurons in vitro.

Intracellular Ca2+ stores were studied in sympathetic neurons grown in primary culture from the superior cervical ganglion of the rat. The [Ca2+]i was measured in single cells using the fluorescent Ca2+ indicator fura-2 and a sensitive microfluorimeter. Superfusion of the cells with 10 mM caffeine elicited a rapid and transient increase in [Ca2+]i in the absence of extracellular Ca2+, indicating the presence of a caffeine-sensitive intracellular Ca2+ storage site. After depletion of the store by mobilization of Ca2+ with caffeine, it could be refilled by elevating [Ca2+]i, allowing multiple caffeine-induced [Ca2+]i transients to be elicited from a single neuron. Ryanodine (1 microM), an alkaloid that promotes Ca2+ release from the sarcoplasmic reticulum, was an effective inhibitor of the caffeine-induced [Ca2+]i transients in sympathetic neurons. Exposure to ryanodine in the presence of caffeine was required to produce a subsequent inhibition of the caffeine-induced response, suggesting a "use-dependent" inhibition that may result from depletion of the Ca2+ stores. In contrast, dantrolene Na (10 microM), an agent known to interfere with Ca2+ release from the sarcoplasmic reticulum, also blocked the caffeine-induced [Ca2+]i transients, but in a time-dependent rather than a use-dependent manner. Electrophysiological measurements using the whole cell version of the patch-clamp technique were made simultaneously with [Ca2+]i microfluorimetric recordings. The magnitude of the [Ca2+]i transients elicited by step depolarizations closely paralleled the magnitude of Ca2+ influx via voltage-sensitive Ca2+ channels, regardless of whether the magnitude of the Ca2+ current was modified by varying the test pulse duration or potential. The relationship between the magnitude of Ca2+ influx and the resulting increase in [Ca2+]i saturated at large Ca2+ influxes resulting from long depolarizations, consistent with the activation of a large capacity, low affinity [Ca2+]i buffering mechanism. Caffeine (10 mM) and ryanodine (10 microM), applied singly or together, produced a small and variable decrease in the [Ca2+]i transient resulting from cell depolarization using the whole-cell patch-clamp technique. We conclude that mammalian sympathetic neurons possess intracellular Ca2+ stores with pharmacological characteristics that closely resemble those found in muscle but that these are relatively small and produce little amplification of [Ca2+]i transients resulting from Ca2+ influx through voltage-sensitive Ca2+ channels.

Dominant role of N-type Ca2+ channels in evoked release of norepinephrine from sympathetic neurons.

Multiple types of calcium channels have been found in neurons, but uncertainty remains about which ones are involved in stimulus-secretion coupling. Two types of calcium channels in rat sympathetic neurons were described, and their relative importance in controlling norepinephrine release was analyzed. N-type and L-type calcium channels differed in voltage dependence, unitary barium conductance, and pharmacology. Nitrendipine inhibited activity of L-type channels but not N-type channels. Potassium-evoked norepinephrine release was markedly reduced by cadmium and the conesnail peptide toxin omega-Conus geographus toxin VIA, agents that block both N- and L-type channels, but was little affected by nitrendipine at concentrations that strongly reduce calcium influx, as measured by fura-2. Thus N-type calcium channels play a dominant role in the depolarization-evoked release of norepinephrine.

Regulation of gastrointestinal function by multiple opioid receptors.

Agonist and antagonist drugs possessing selectivity for individual types of opioid receptors have been employed in vitro and in vivo to determine the mechanisms by which opioids regulate gastrointestinal functions. Selective mu opioid agonists given by intracerebroventricular (i.c.v.) injection, by intrathecal (i.t.) injection, or by peripheral (s.c. or i.v.) injection in rats or mice decreased gastrointestinal transit and motility, inhibited gastric secretion, and suppressed experimentally-induced diarrhea. Selective delta agonists, by contrast, inhibited gastrointestinal transit after i.t., but not after i.c.v. or s.c. administration. Delta agonists also did not alter gastric secretion after i.c.v. or s.c. injection. However, delta agonists exhibited antidiarrheal effects after i.c.v., i.t., or s.c. administration. Kappa agonists given i.c.v. had no effect on gastrointestinal transit in rats or mice or on gastric secretion in rats, but exhibited antidiarrheal effects in mice. The kappa agonist U-50, 488H given peripherally increased gastric acid secretion. Different types of opioid receptors in different anatomical sites influence differently gastrointestinal motility and propulsion, gastric secretion, and mucosal transport. Brain, spinal cord, enteric neural and smooth muscle opioid receptors represent chemosensitive sites for regulation of gastrointestinal function.

Distribution of multiple types of Ca2+ channels in rat sympathetic neurons in vitro.

Ca2+ influx through voltage sensitive Ca2+ channels produces a rise in intracellular-free Ca2+, [Ca2+]i, that serves as a trigger for the release of neurotransmitters. We measured [Ca2+]i in primary cultures of superior cervical ganglion (SCG) neurons of the rat using 2-(6-(bis(carboxymethyl)amino)-5-methylphenoxy)ethoxy-2-benzofuranyl)5- oxazole carboxylic acid-based microfluorimetry. Recordings were obtained from either single or small bundles of neuronal processes and compared with recordings from single neuronal cell bodies. Depolarization with 50 mM K+ produced a rapid increase in [Ca2+]i consisting of both transient and sustained components. This response pattern was seen in recordings from both the soma and processes of SCG neurons. The entire response could be reversibly blocked by 30 microM La3+. Nitrendipine, 1 microM, inhibited the response by 52 +/- 7% and 49 +/- 7% in the soma and processes, respectively. The dihydropyridine (DHP) agonist 1,4-dihydro-2,-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine- 5-carboxylic acid methyl ester enhanced depolarization-induced increases in [Ca2+]i in both regions of the neuron. The transient component of the response was greatly reduced by predepolarization, and the remaining sustained component was inhibited 77 +/- 7% by nitrendipine (1 microM). These data demonstrate that both DHP-sensitive and -insensitive Ca2+ channels are present in processes as well as cell bodies of SCG neurons. The importance of these findings is discussed in relation to the insensitivity of neurotransmitter release from sympathetic neurons to DHP antagonists.

Down regulation of protein kinase C in neuronal cells: effects on neurotransmitter release.

We investigated the effects of phorbol esters on protein kinase C (PKC) activity and on neurotransmitter release from cultured neuronal cells. Both differentiated and undifferentiated PC12 pheochromocytoma cells contained high levels of protein PKC. Under normal conditions all the enzyme activity was found in the cytoplasm. Addition of the phorbol esters phorbol 12-myristate-13-acetate (TPA) or phorbol 12,13-dibutyrate (PDBu) caused a rapid translocation of PKC from the cytoplasm to the particulate fraction. Continued culture of cells with these phorbol esters resulted in the decline of total PKC activity. After 10-20 hr of culture, both membrane and cytoplasmic PKC activity had declined to background levels. cAMP-dependent and Ca2+/calmodulin-dependent protein kinase activities were only slightly affected by chronic phorbol ester treatment. Addition of active phorbol esters to PC12 cells produced an enhancement of the depolarization-induced release of 3H-norepinephrine. Following chronic phorbol ester treatment, the ability of these substances to enhance evoked catecholamine release was lost. Furthermore, depolarizing stimuli released considerably less 3H-norepinephrine than in control untreated cells. Phorbol esters also enhanced depolarization-induced 3H-norepinephrine release from primary cultures of rat sympathetic neurons. Chronic treatment of these neurons with phorbol esters also resulted in the loss of their ability to enhance transmitter release and in a large reduction in the extent of depolarization-evoked transmitter release. Chronic phorbol ester treatment also resulted in the disappearance of PKC from sympathetic neurons, but had little effect on cAMP-dependent or Ca2+/calmodulin-dependent kinase activities. These results demonstrate that PKC-deficient neurons can be prepared. The data also demonstrate that depolarization-induced neurotransmitter release is mediated by both protein kinase C-dependent and independent pathways.

Pharmacologic evaluation of a cyclic somatostatin analog with antagonist activity at mu opioid receptors in vitro.

The cyclic somatostatin analog D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2(CTP) was evaluated for agonist and opioid antagonist actions and receptor selectivity in two bioassays: electrically stimulated guinea pig isolated ileum (GPI) and mouse isolated vas deferens (MVD). CTP (100, 300, 1000 nM) produced concentration-dependent antagonism of the mu agonist [Me-Phe3,D-Pro4]morphiceptin (PL017) in both the GPI and MVD. Schild analysis of the interactions between CTP and PL017 indicated competitive antagonism between these peptides (Schild slope GPI -0.97 +/- 0.16, Schild slope MVD -1.4 +/- 0.4), and also suggested that the mu receptors in the two tissues are not different (pA2 GPI 7.1 +/- 0.17, pA2 MVD 6.9 +/- 0.16). The effects of the delta selective agonist [D-Pen2,D-Pen5]enkephalin in the MVD were not antagonized by CTP. Likewise, in the GPI, CTP did not antagonize the kappa agonist (trans-3-4-dichloro-N-methyl-N-(2-(1-pyrrolidinyl)cyclohexyl)benzenea cetamine (U50, 488H). In comparison, naloxone antagonized both PL017 and U50,488H in the GPI, as well as [D-Pen2,D-Pen5]enkephalin and PL017 in the MVD. In the MVD, CTP also exerted weak somatostatin-like actions (35% maximal inhibition) that could not be demonstrated in somatostatin-tolerant tissues. It also showed inhibitory actions at very high concentrations (3000 and 10,000 nM) that were blocked by both naloxone and the delta antagonist N,N-diallyl-Tyr-AIB-AIB-Phe-Leu-OH (ICI 174,864). ICI 174,864 antagonized [D-Pen2,D-Pen5]enkephalin in the MVD, but did not affect PL017. These results indicate that CTP is a selective mu receptor antagonist in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple calcium channels mediate neurotransmitter release from peripheral neurons.

We examined the effects of dihydropyridine drugs on evoked neurotransmitter release from cultured neonatal rat sensory and sympathetic neurons. Depolarization with K+-rich solutions increased the release of substance P from cultured sensory neurons. This release was enhanced by BAY K8644 and (+)-202791 and was blocked by a variety of other dihydropyridines including (-)-202791, by Co2+, or in Ca2+-free solutions. K+-rich solutions also stimulated the release of [3H]norepinephrine from cultured sympathetic neurons. This release was also completely blocked by Co2+ or in Ca2+-free solution. In contrast to the situation in sensory neurons, however, the evoked release of [3H]norepinephrine was completely resistant to the blocking effects of dihydropyridine such as nimodipine. However, BAY K8644 was able to enhance the evoked release of [3H]norepinephrine, and this enhancement was blocked by nimodipine. These results are discussed in relation to the possible participation of multiple types of calcium channels in the release of neurotransmitters.

Neurogenic mechanism of action of motilin in the canine isolated small intestine ex vivo.

The contractile effects of the brain gut peptide motilin were evaluated in isolated, vascularly perfused canine small intestine segments. Motilin (0.04-5.0 micrograms) produced dose-related increases in intraluminal pressure after bolus intraarterial injection. The contractile activity was effectively antagonized by vascular perfusion with either tetrodotoxin (100 ng/ml) or atropine (40 ng/ml), indicating that the primary site of action of motilin was on cholinergic neural elements of the enteric nervous system. Responses to motilin were partially inhibited by hexamethonium perfusion (15 micrograms/ml) suggesting that motilin acts, in part, on preganglionic neural elements proximal to nicotinic cholinergic synapses within the enteric nervous system. No evidence for a direct action of motilin on the smooth muscle of the intestine was observed. Perfusion of the segments with naloxone (up to 3.0 micrograms/ml) did not affect the responses of the bowel to motilin, suggesting that opioid systems are not directly involved in the contractile responses. These data indicate that motilin acts predominantly on cholinergic nerves of the enteric plexes to stimulate contractions of the intestine. In addition, motilin acts, in part, on the preganglionic interneurons of the enteric nervous system and therefore requires intact ganglionic connections. Vascular perfusion with motilin (5-50 ng/ml) also produced dose-related increases in intraluminal pressure suggesting that motilin may act through the circulation in vivo to stimulate intestinal motility. However, these concentrations are significantly greater than those measured during contractile activity in vivo.

Conformationally restricted cyclic analogues of substance P: insight into the receptor binding process.

Three new cyclic substance P analogues were prepared to examine the possible role of a pseudocyclic turn structure for receptor recognition. In the guinea pig isolated ileum [Cys5, Cys11]-SP5-11-NH2 and [Cys6, Cys11]-SP5-11-NH2 were inactive at concentrations up to 100 microM, while [Cys5, Cys6, Nle11]-SP was a weak agonist. The order of relative affinities on the rat brain radioreceptor assay was as follows: [Cys5, Cys6, Nle11]-SP greater than [Cys5, Cys11]-SP5-11-NH2 greater than [Cys6, Cys11]-SP5-11-NH2. We interpret these results to indicate that a pseudocyclic structure of the 5-11 sequence may not be an important factor involved in the receptor recognition of substance P.

Studies in vitro with ICI 174,864, [D-Pen2, D-Pen5]-enkephalin (DPDPE) and [D-Ala2, NMePhe4, Gly-ol]-enkephalin (DAGO).

The interactions of a proposed, selective delta receptor antagonist (ICI 174,864) and selective agonists at mu and delta receptors, [D-Ala2, NMePhe4, Gly-ol]-enkephalin (DAGO) and [D-Pen2, D-Pen5]-enkephalin (DPDPE), respectively, have been studied using the electrically-stimulated mouse isolated vas deferens (MVD) and the guinea-pig isolated ileum (GPI). Incubation of increasing concentrations of ICI 174,864 (10,30,100 and 300 nM) produced a dose-related and parallel rightward displacement of the DPDPE dose-response curve in the MVD. In contrast, ICI 174,864 (300-3000 nM) failed to affect the DAGO dose-response curve in the same tissue. Analysis of the DPDPE-ICI 174,864 interaction in the MVD using the pA2 method revealed a Schild plot slope of -0.68 suggesting the involvement of more than one population of receptors. ICI 174,864 (300 nM) failed to antagonize DPDPE in the GPI at doses up to 30 microM. These results suggest that (a) ICI 174,864 acts as a selective delta antagonist in the MVD; (b) DPDPE interacts with mu receptors in the MVD but only at very high concentrations, and (c) delta receptors appear not to be of functional importance in the GPI.

Mu, but not kappa, opioid agonists induce contractions of the canine small intestine ex vivo.

The proposed kappa opioid receptor agonists ethylketocyclazocine (EK), nalorphine, bremazocine and U-50,488H were evaluated for their ability to produce contractions of isolated, vascularly perfused canine small intestinal segments. Responses to these agonists were compared to those of morphine and phenazocine, a mu benzomorphan. Morphine (0.04-25 micrograms) and phenazocine (0.01-3.0 micrograms) both produced naloxone-reversible contractions, suggesting that the responses were mediated largely by mu opioid receptors. In contrast, the proposed kappa agonists were ineffective in producing intestinal stimulation, with only EK (1-100 micrograms) showing minimal but significant activity at very high doses. We suggest that the effects of EK may be mediated through mu opioid receptors and that kappa receptors appear not to be involved in the contractile response of the dog small intestine to opioids.