Interaction of gastrin I, secretin, and cholecystokinin on gallbladder smooth muscle.

The effect of cholecystokinin (CCK), the octapeptide of cholecystokinin (CCK-OP), gastrin I, and secretin was studied on guinea pig gallbladder smooth muscle in vitro. Both CCK and CCK-OP stimulated gallbladder contraction, with CCK-OP being more potent. Gastrin I, over a wide dose range, had no effect on gallbladder contractility. Secretin alone also showed no effect on gallbladder smooth muscle but in combination with CCK-OP it produced a noncompetitive type of inhibition. Michaelis-Menten kinetics showed the calculated maximum response of the secretin plus CCK-OP interaction to be less than with CCK-OP alone. There was no change in the dose required to achieve half-maximal response, D50. These studies indicate that: a) CCK-OP has a greater effect on gallbladder contractility than CCK, b) gastrin I has no effect on gallbladder muscle tone, and c) secretin acts as a noncompetitive antagonist of CCK-OP. These findings suggest that gallbladder motor function may be determined in part by the interaction of secretin and CCK rather than solely in response to CCK.     

Receptors on smooth muscle cells: characterization by contraction and specific antagonists

Smooth muscle cells were isolated from the stomach of the guinea pig, and the kinetics, stoichiometry, and specificity of contraction in response to the C-terminal octapeptides of cholecystokinin (CCK-OP), gastrin-17, and acetylcholine were examined. All three agonists elicited dose-dependent peak contraction that did not depend on the presence of extra-cellular calcium. The potencies of CCK-OP and gastrin-17 were equal (D50, 10(-11) M) and 10 times greater than the potency of acetylcholine (D50, 10(-10) M). A combination of low doses of acetylcholine and CCK-OP was synergistic; however, its effect did not exceed the maximal responses to either agonists alone or to high extracellular concentrations of calcium. The specificity of the receptors was established by the use of atropine and the two CCK-receptor antagonists dibutyryl cGMP and proglumide. The span of the dose-response curves was wide, suggesting the existence of receptor heterogeneity. It is concluded that gastric smooth muscle cells of the guinea pig possess distinct, high-affinity receptors for CCK-gastrin and acetylcholine; the receptors mediate contraction that is not immediately dependent on the presence of extracellular calcium.     

Age-related changes in gallbladder contractility and gallbladder cholecystokinin receptor population in the guinea pig

We have examined the effects of aging on guinea pig biliary motility both in vitro and in vivo. The first experiment compared contractile tension of gallbladder strips from young adult (6-12 months old) and 3-year-old guinea pigs in vitro. Contraction of gallbladder strips from the young guinea pigs was twice as forceful and was more sensitive to octapeptide of cholecystokinin (CCK-8) stimulation than the gallbladder strips from the older guinea pigs. The two groups were also studied in vivo by measuring changes in the intraluminal pressure of the gallbladder in response to exogenously administered doses of CCK-8. Young adult guinea pigs were more sensitive to CCK-8 at the lower doses tested and demonstrated gallbladder contractions that were more forceful than that of the old guinea pigs. CCK receptors were measured on gallbladder muscularis membranes from young adult and old guinea pigs. The number of receptors on gallbladder membranes decreased with age: 65.0 +/- 17.7 fmoles/mg protein on membranes from 1 year old; 7.9 +/- 2.0 fmoles/mg protein on membranes from 3 years old. The binding affinity of CCK receptors on gallbladder muscularis membranes for binding to CCK-8 was not significantly different in the two age groups studied. We conclude that age-related decreases in gallbladder responses to CCK-8 may be due to decreased concentrations of CCK receptors on gallbladder muscle cells.     

Simultaneous release of substance P- and calcitonin gene-related peptide (CGRP)-like immunoreactivity from isolated muscle of the guinea pig urinary bladder

Capsaicin (10 microM) induced a tetrodotoxin (TTX)-resistant release of substance P (SP)- and calcitonin gene-related peptide (CGRP)-like immunoreactivity (LI) from muscle strips of the guinea pig isolated urinary bladder. A second application of capsaicin had no effect, indicating a specific effect on sensory nerves (desensitization). In functional experiments, capsaicin produced a phasic contraction of isolated bladder strips. This response was TTX-resistant, exhibited desensitization and was specifically antagonized by [D-Pro4, D-Trp7.9, Phe11] SP(4-11) a SP antagonist which also reduced, at a similar extent, the contraction induced by exogenous SP. These findings provide direct neurochemical and functional evidence for a transmitter role for a SP-like peptide(s) from peripheral sensory terminals in the guinea pig urinary bladder.     

Effect of cholecystokinin-octapeptide on opossum lower esophageal sphincter

We evaluated the action of cholecystokini-octapeptide (CCK-OP) on lower esophageal sphincter (LES) pressure in the opossum. LES pressure was recorded by an infused sleeve device that straddled the sphincter, whereas intraluminal esophageal pressure and gastric pressure were recorded via conventional manometric catheters. Progressive intravenous pulse doses of CCK-OP caused 1) graded increases in LES pressure, 2) circular and longitudinal smooth muscle contraction in esophageal body, and 3) mild increases in intragastric pressure. Pressor effect of CCK-OP on the LES was weakly antagonized by tetrodotoxin (TTX), but not by atropine, phentolamine, or pyrilamine. TTX antagonism of CCK-OP appeared to be nonspecific because TTX also partially antagonized LES contractions induced by pentagastrin, substance P, and bethanechol. We conclude that CCK-OP at doses that cause LES relaxation in other species induces LES contraction in the opossum. This pressor effect appears to be elicited by a direct action of the hormone on LES smooth muscle.     

Actions of peptides isolated from amphibian skin on pancreatic acinar cells.

In dispersed acinar cells prepared from guinea pig pancreas, peptides isolated from amphibian skin (caerulein, bombesin, litorin, and physalaemin) as well as eledoisin, a peptide isolated from the posterior salivary gland of a Mediterranean octopod, increased outflux of 45Ca, release of bound 45Ca, accumulation of cyclic GMP, and release of amylase. In addition, bombesin, litorin, physalaemin, and eledoisin each increased the initial uptake of 45Ca by dispersed acinar cells, whereas C-terminal octapeptide of porcine cholecystokinin (CCK-OP) and carbamylcholine did not increase the initial uptake of 45Ca but, rather, abolished the increase caused by the other agents. None of the actions of these amphibian peptides was altered by concentrations of atropine sufficient to abolish the effects of muscarinic cholinergic agents. None of the amphibian peptides altered cellular cyclic AMP or the increase caused by secretin or porcine vasoactive intestinal peptide (VIP). Acinar cells preincubated with 45Ca plus bombesin showed the same rate of release of 45Ca as did control cells and this rate was not altered by adding bombesin but was increased fivefold by adding CCK-OP. In terms of their chemical structures as well as the potency and efficacy with which they alter acinar cell function, the amphibian peptides plus CCK-OP can be grouped into three pairs: caerulein with CCK-OP, bombesin with litorin, and physalaemin with eledoisin.     

Highly 4-aminopyridine sensitive delayed rectifier current modulates the excitability of guinea pig cerebellar Purkinje cells

The effects of low concentrations of 4-aminopyridine (4-AP) on the membrane properties of guinea pig cerebellar Purkinje cells were investigated in slice preparation using intracellular recordings. It was found that 1–10 μM 4-AP did not affect the resting potential or the input resistance of the cells, but reduced markedly the duration of the slowly depolarizing potential (SDP), and thus the latency to the firing of Ca²⁺ spikes in response to intracellular current pulses. Intradendritic recordings in the presence of tetrodotoxin, Cd²⁺, and low [Ca²⁺]_o, which blocked all the regenerative responses, exhibited prominent membrane outward rectification in response to depolarizing current pulses. Under these conditions, the SDP was abolished and, in contrast, a slowly developing hyperpolarization was consistently observed. Application of 10 μM 4-AP reduced the outward membrane rectification in a reversible manner, but did not affect the transient hyperpolarization, which is usually attributed to the activation of potassium "A" current. These results demonstrate, for the first time, the presence of a highly 4-AP sensitive delayed rectifier in guinea pig cerebellar Purkinje cells, which prominently affects their excitability. The results also indicate that the slowly depolarizing potential of guinea pig Purkinje cells does not involve inactivation of transient potassium currents, which has been suggested previously as an underlying mechanism for this phenomenon in turtle Purkinje cells. Electronic Publication     

Enhanced fluid transport across gallbladder mucosa in experimental cholelithiasis.

Fluid transport of the gallbladder has been studied in two models of experimental cholelithiasis: dihydrocholesterol-induced gallstones in the rabbit and lincomycin-induced gallstones in the guinea pig. Using the noneverted explained gallbladder of the rabbit and the guinea pig, the transport of luminal to serosal fluid has been quantitated before, during, and after stone formation. The everted gallbladder preparation of the rabbit has also been used to measure fluid transport before and during gallstone formation. In both models, an increased fluid transport was observed in the phase of gallstone induction and a return to normal after stones were formed. This abnormality preceded the appearance of conventional histological features of cholecystitis. There was also a coincidental increase in glycoprotein production from and cell proliferation of the gallbladder epithelium. This enhancement of fluid transfer may play a contributing role in the genesis of gallstones.     

Neurochemical evidence for the involvement of N-type calcium channels in transmitter secretion from peripheral endings of sensory nerves in guinea pigs.

In the guinea pig ureter, substance P-(SP) and calcitonin gene-related peptide-(CGRP) like immunoreactivity (LI) were depleted by systemic capsaicin pretreatment, indicating that they are entirely stored in peripheral endings of primary afferent neurons. Electrical field stimulation (20 Hz, 60 V, 0.5 ms) evoked the simultaneous release of SP- and CGRP-LI from superfused guinea pig ureters which was abolished by tetrodotoxin (0.3 microM). omega-Conotoxin (0.1 microM), a potent blocker of N-type voltage-sensitive calcium channels, reduced by 50-70% the evoked release of both peptides. These findings provide direct neurochemical evidence indicating that conotoxin-sensitive calcium channels play a role in transmitter secretion evoked by antidromic invasion of peripheral terminals of capsaicin-sensitive primary afferents.     

Cholecystokinin octapeptide and gastric mechanoreceptor activity in rat brain

Extracellular unitary recordings were made from neurons in the rat dorsal vagal nucleus, and the response of these neurons to iontophoretically applied cholecystokinin octapeptide (CCK-OP) and proglumide (a novel CCK antagonist) was studied. The effect of CCK-OP and proglumide on the response of the neurons to gastric distension was also studied. Seventy-four percent (n = 39) of the neurons studied were excited by CCK-OP. It was not possible to antagonize the response to CCK-OP with proglumide. CCK-OP was also found to be an effective modulator of the response to gastric distension; the effects were diverse ("on" responses were augmented; "off" and "transient" responses were diminished) but were always the same within each characteristic pattern of response to distension. These results provide supporting evidence for CCK-OP, a peptide common to both brain and gut, acting in the role of a neural modulator in the control of the gastrointestinal tract. It is possible to speculate that the potent effect of CCK-OP on neuronal excitability and on the central representation of gastric mechanoreceptor activity may well substantiate the role of CCK-OP as a central regulator of feeding behavior.     

Effects of neurotransmitter release on mucosal transport in guinea pig ileum

Scorpion venom (Leiurus quinquestriatus), a substance that evokes neurotransmitter release by depolarizing neurons, was used to activate enteric neurons in short-circuited guinea pig ileum. Scorpion venom increased transmural potential difference and short-circuit current, and this response was similar to the increase that occurred after electrical stimulation of enteric neurons. The stimulus- or venom-evoked response in short-circuit current was abolished by tetrodotoxin. Atropine reduced by 47% the increments in short-circuit current produced by either electrical stimulation or venom. Scorpion venom increased active chloride secretion in short-circuited guinea pig ileal mucosa but had no significant effect on active sodium absorption, residual flux, or total tissue conductance. No morphological changes in transmission electron micrographs of ileal mucosa treated with scorpion venom were evident compared with controls. Alanine caused an increase in short-circuit current in venom-treated tissue that was similar to control values. These results show that scorpion venom mimics the mucosal effects of electrical activation of enteric neurons. These results suggest that a significant component of both scorpion venom action and the response to electrical field stimulation is mediated by neural release of acetylcholine, which activates epithelial muscarinic receptors.     

Isoproterenol-induced restoration of contraction in K+-depolarized hearts: relationship to cAMP

The action of isoproterenol on inotropic state, cAMP concentration, and phosphorylase b-to-a conversion was studied under conditions that are known to alter membrane properties in guinea pig papillary muscles. In accord with the results of other investigators, contractile events induced by electrical stimulation were abolished by tetrodotoxin (10 microM) or 22 mM K+ and were subsequently restored by the addition of isoproterenol (10 nM-100 microM). However, in the presence of 22 mM K+, but not tetrodotoxin, the dose-response and temporal relationships between isoproterenol and elevations in cAMP concentration were shifted to the right, whereas those for phosphorylase activation were shifted to the left. Thus the low concentrations (less than 10 nM) of isoproterenol that restored tension development did not produce a measurable increase in cAMP. Contractile responses induced by 10 nM isoproterenol were blocked by (--)-propranolol but not by (+)-propranolol or phentolamine. Methoxamine did not restore contractile events in 22 mM K+-treated muscles or induce changes in cAMP content or phosphorylase activation. These results show that conditions can be obtained (e.g., partial depolarization of cardiac cell membranes) in which beta-adrenergic receptor activation leads to restoration of inotropic state in guinea pig myocardium without an obligatory increase in tissue cAMP content. The restoration of contractile events and enhancement of phosphorylase response under these conditions suggest that beta-adrenergic receptor activation may lead to Ca2+-channel activation directly or by increasing a small pool of cAMP and thereby altering localized protein phosphorylation in the cell membrane.     

Non-neuronal excitatory neurotensin receptors on the taenia coli of the guinea pig: lack of influence of tetrodotoxin and dynorphin

Picomoles of neurotensin caused contractions of the smooth muscles of the taenia coli and the myenteric plexus preparation of the guinea pig ileum. The musculotropic effect of neurotensin on the taenia coli was resistant to tetrodotoxin and to treatment with dynorphin. In contrast, the contractile activity of the neuropeptide in the ileum was substantially modified by pretreatment with tetrodotoxin or dynorphin. It is concluded that, whereas neurotensin causes a direct muscular response on the taenia coli via activation of selective peptide receptors probably located on the muscle membrane, the neuropeptide causes a predominant indirect action on the ileum mediated apparently via the release of acetylcholine from the nerve terminals of the myenteric plexus. Neurotensin may play a role in the control of gut peristalis either by modifying the release of neurotransmitters or by directly influencing the smooth muscles of the intestines.     

Intraluminal prostaglandin E2 affects gallbladder function by activation of intramural nerves in the anaesthetized cat

Gallbladder mucosal net fluid transport and motility were measured in vivo by a continuous perfusion technique in the anaesthetized cat. Prostaglandin E2, administered to the perfused gallbladder lumen, caused a contraction decreasing gallbladder volume capacity, and induced a secretory response by the mucosa. These effects by prostaglandin E2 were abolished by the nerve-blocking agent tetrodotoxin (administered close intraarterially) and somatostatin (administered intravenously), but not by intravenous hexamethonium. Atropine (administered intravenously) reduced the order of magnitude of the gallbladder contraction in response to prostaglandin E2 but did not affect the secretory response by the mucosa. Neither of these drugs significantly affected gallbladder volume capacity or mucosal fluid transport during basal conditions. Tetrodotoxin did not abolish the gallbladder responses to intravenous cholecystokinin or vasoactive intestinal peptide, peptides known to act directly upon smooth muscle and epithelial cell receptors, respectively. It is suggested that prostaglandin E2 affects gallbladder function in vivo mainly by activation of postganglionic non-cholinergic intramural nerve cells.     

The presence of N-methyl-D-aspartate-type receptors for glutamic acid in the guinea pig myenteric plexus

The actions of agonists and antagonists of excitatory amino acid receptors were studied in the isolated ileal longitudinal muscle-myenteric plexus preparation of the guinea pig incubated 'in vitro', by recording the contraction of the longitudinal muscle. L-Glutamate, L-aspartate, quinolinate and N-methyl-D-aspartate (NMDA), in concentrations ranging from 10(-6) to 10(-4) M, induced a rapid contraction of this preparation while kainate and quisqualate were not active at a concentration of 10(-4) M. The excitatory amino acid responses were competitively antagonized by 2-amino-5-phosphonovalerate. They were also prevented by Mg2+ ions (0.1-1 mM), by tetrodotoxin 3 X 10(-6) M and by hyoscine 10(-7) M. The last observations suggest that the myenteric cholinergic interneurons are in some way involved in the glutamate-induced ileal contraction. These results demonstrate the existence of receptors for excitatory amino acids (possibly of NMDA type) in the myenteric plexus of the guinea pig.     

Biologic activity of guinea pig IFN-gamma in vitro.

Interferon-gamma (IFN-gamma) plays a key role in the induction and maintenance of immunity against intracellular infectious agents. Compared to other species, little is known about the biology of this cytokine in the guinea pig (Cavia porcellus). We found that in contrast to humans and mice, IFN-gamma in the guinea pig did not induce the antiviral state, which in other species leads to protection of IFN-gamma -stimulated fibroblasts from the cytopathic effect (CPE) of subsequent viral infections. As an alternative strategy to detect and quantify guinea pig IFN-gamma activity in vitro, a reporter system using guinea pig fibroblasts transfected with a luciferase gene, which is regulated by an IFN-stimulated response element (ISRE), was established. With the help of the highly sensitive reporter assay system, the biologic activity of recombinant guinea pig IFN-gamma (GpIFN-gamma, from prokaryotic and eukaryotic expression systems was detected. The response to both native and recombinant GpIFN-gamma was inhibited by a rabbit antiserum directed against the recombinant cytokine expressed in Escherichia coli, demonstrating structural and functional homology of native and recombinant GpIFN-gamma. Stimulation with GpIFN-gamma, obtained from transfected cells, induced upregulation of MHC class I expression in a guinea pig fibroblast line. The restricted activity of GpIFN-gamma might have implications for this species' ability to control infections with intracellular pathogens.     

Interaction of acetylcholine and cholecystokinin with dispersed smooth muscle cells.

Isolated gastric smooth muscle cells were prepared from the stomach of Bufo marinus by successive incubation in collagenase without added trypsin. Contraction was determined by image-splitting micrometry and expressed as the mean percentage decrease in cell length from control. Peak contractile response was attained within 30 s. Dose-response curves constructed from peak responses showed that the maximal responses to CCK-OP (37.2 +/- 3.8%), acetylcholine (35.3 +/- 2.5%), and Ca2+ (42.3 +/- 0.9%) were similar. The D50s for octapeptide of cholecystokinin (CCK-OP) and acetylcholine were around 10(-12) M and 10(-11) M, respectively. The response to a combination of submaximal concentrations of acetylcholine and CCK-OP exceeded the individual responses but did not exceed the maximal response to either agent alone. A low concentration of atropine (5 X 10(-10) M) inhibited specifically the maximal response to acetylcholine. A high concentration of atropine (5 X 10(-8) M) inhibited partially the maximal response to CCK-OP but had no effect on the maximal response to Ca2+. It was concluded that 1) dispersed gastric smooth muscle cells are highly sensitive to stimulation; 2) CCK-OP has a direct (myogenic) contractile effect on gastric smooth muscle; and 3) the effect of CCK-OP and acetylcholine are mediated by separate receptors.     

Efffect of vasoactive intestinal polypeptide on gallbladder smooth muscle in vitro

The effect of vasoactive intestinal polypeptide (VIP) on basal and octapeptide of cholecystokinin (OP-CCK) induced tension was examined with guinea pig gallbladder smooth muscle strips in vitro. VIP alone produced dose-related decreases in resting tension and antagonized spontaneous contractile activity where present. In combination with OP-CCK, VIP decreased the expected contractile respone. The degree of antagonism depended upon the concentrations of OP-CCK and VIP. VIP had no effect on acetylcholine-induced contractions. From these observations, we propose that VIP can affect gallbladder motor activity by decreaseing smooth muscle tone and by antagonizing cholecystokinin. These findings lend further support to our proposal that gallbladder motor function may depend upon the action and interaction of the gastrointestinal hormones.     

Production, assay and partial characterization of guinea pig interleukin 2

Optimum conditions for the production and assay of guinea pig interleukin-2 (IL-2) have been established. The mitogenic activities of serial dilutions of guinea pig IL-2 preparations were compared in cultures of guinea pig peripheral blood lymphocytes prestimulated for 7 days with phytohemagglutinin (PHA) used at 1 microgram/ml. Parallel log dose-log response curves were used for quantitative comparisons. Optimum IL-2 yields were obtained from cultures of lymph node lymphocytes stimulated for 20 h with Concanavalin A (ConA) at 5 micrograms/ml. Guinea pig T cell lines reactive to mycobacterial antigens were propagated for several months using our IL-2 preparations. The molecular weight of guinea pig IL-2 was estimated to be 30,000 using S-200 gel filtration. The species specificities of guinea pig, human, mouse and rat IL-2s were examined. It was shown that guinea pig T lymphocyte blasts were stimulated only weakly with human IL-2 and not at all with mouse and rat IL-2.     

Studies on the electrical stimulation-induced contractile responses of hamster and mouse gallbladders.

Isolated gallbladders of both hamsters and mice showed a contractile response consisting of initial and secondary contractile responses to electrical stimulation with rectangular pulses (50 volt, 40 Hz) of 6 msec duration for a period of 10 seconds. The duration time of the contractile response was very long. This was because the duration time of the process of the initial contractile response was as short as 5 to 40 seconds, but that of the secondary contractile response was as long as 8 to 15 minutes. Both initial and secondary contractile responses of the gallbladder in both hamsters and mice were little affected by atropine (1 x 10(-6)M, guanethidine (1 x 10(-6) M) and tetrodotoxin (1 x 10(-7) M). In both animals, further, the initial contractile response of the gallbladder was little affected by indomethacin (1 x 10(-5) M) and 5, 8, 11, 14-eicosatetraynoic acid (ETYA) (5 x 10(-6) g/ml), while the amplitude of the secondary contractile response of the gallbladder was significantly reduced by such drugs. From these results, we obtain the following conclusions: 1) In both animals, the intramural nerves of the gallbladder may have no influence on the both initial and secondary contractile responses of the gallbladder. 2) The initial contractile response is probably mediated by a direct effect of the electrical stimulation to the muscle in the wall of the gallbladder, while the secondary contractile response is probably attributable to prostaglandins released from the wall of the gallbladder by a direct effect of the electrical stimulation to the wall.