Enteric motor neurons form synaptic-like junctions with interstitial cells of Cajal in the canine gastric antrum

Morphological studies have shown synaptic-like structures between enteric nerve terminals and interstitial cells of Cajal (ICC) in mouse and guinea pig gastrointestinal tracts. Functional studies of mice lacking certain classes of ICC have also suggested that ICC mediate enteric motor neurotransmission. We have performed morphological experiments to determine the relationship between enteric nerves and ICC in the canine gastric antrum with the hypothesis that conservation of morphological features may indicate similar functional roles for ICC in mice and thicker-walled gastrointestinal organs of larger mammals. Four classes of ICC were identified based on anatomical location within the tunica muscularis. ICC in the myenteric plexus region (IC-MY) formed a network of cells that were interconnected to each other and to smooth muscle cells by gap junctions. Intramuscular interstitial cells (IC-IM) were found in muscle bundles of the circular and longitudinal layers. ICC were located along septa (IC-SEP) that separated the circular muscle into bundles and were also located along the submucosal surface of the circular muscle layer (IC-SM). Immunohistochemistry revealed close physical associations between excitatory and inhibitory nerve fibers and ICC. These contacts were synaptic-like with pre- and postjunctional electron-dense regions. Synaptic-like contacts between enteric neurons and smooth muscle cells were never observed. Innervated ICC formed gap junctions with neighboring smooth muscle cells. These data show that ICC in the canine stomach are innervated by enteric neurons and express similar structural features to innervated ICC in the murine GI tract. This morphology implies similar functional roles for ICC in this species.     

A STUDY OF THE INNERVATION OF THE TAENIA COLI

An electrophysiological and anatomical study of the guinea pig taenia coli is reported. Changing the membrane potential of single cells cannot modulate the rate of firing action potentials but does reveal electrical coupling between the cells during propagation. The amplitude of the junction potentials which occur during transmission from inhibitory nerves is unaffected in many cells during alteration of the membrane potential, indicating electrical coupling during transmission. The taenia coli is shown to consist of smooth muscle bundles which anastomose. There are tight junctions between the cells in the bundles, and these probably provide the pathway for the electrical coupling. The smooth muscle cells towards the serosal surface of the taenia coli are shown electrophysiologically to have an extensive intramural inhibitory innervation, but a sparse sympathetic inhibitory and cholinergic excitatory innervation. These results are in accordance with the distribution of these nerves as determined histochemically. As single axons are only rarely observed in the taenia coli, it is suggested that the only muscle cells which undergo permeability changes during transmission are those adjacent to varicosities in the nerve bundles. The remaining muscle cells then undergo potential changes during transmission because of electrical coupling through the tight junctions.     

Evoked secretion of [3H]noradrenaline and ATP from nerve varicosities isolated from the myenteric plexus of the guinea pig ileum

Neuronal varicosities, isolated from the myenteric plexus of guinea pig ileum longitudinal muscle, were incubated with [3H]noradrenaline to label the contents of the noradrenergic secretory vesicles. Exposure of these varicosities to KCl, nicotine, or acetylcholine resulted in the Ca2+ -dependent release of [3H]noradrenaline. Veratridine also evoked a large efflux of [3H] from this preparation, but this release was only partially Ca2+ dependent. The alpha 2-adrenoceptor agonist, clonidine, inhibited the K+-, nicotine-, and acetylcholine-induced release of [3H]noradrenaline. Similarly, exogenously administered (-)noradrenaline was an effective inhibitor of the K+ -evoked release of [3H]noradrenaline. The alpha 2-adrenoceptor antagonist, yohimbine, antagonized the inhibitory actions of both clonidine and (-)noradrenaline on the K+ -evoked release of [3H]noradrenaline from myenteric varicosities. Nicotine, acetylcholine, KCl, and veratridine also released ATP from these guinea pig ileal myenteric varicosities. However, the evoked release of ATP was unaffected by clonidine. These results indicate that myenteric varicosities can take up and release [3H]noradrenaline and that they possess presynaptic alpha 2-adrenoceptors which, when activated, inhibit the release of [3H]noradrenaline. These receptors may play a role in modulating the release of noradrenaline in the myenteric plexus in vivo. In addition, the present results suggest that ATP and [3H]noradrenaline may not be released from the same population of secretory vesicles in neuronal varicosities isolated from guinea pig ileum longitudinal muscle.     

Targeting the restricted α-subunit repertoire of airway smooth muscle GABAA receptors augments airway smooth muscle relaxation

The prevalence of asthma has taken on pandemic proportions. Since this disease predisposes patients to severe acute airway constriction, novel mechanisms capable of promoting airway smooth muscle relaxation would be clinically valuable. We have recently demonstrated that activation of endogenous airway smooth muscle GABA(A) receptors potentiates β-adrenoceptor-mediated relaxation, and molecular analysis of airway smooth muscle reveals that the α-subunit component of these GABA(A) receptors is limited to the α(4)- and α(5)-subunits. We questioned whether ligands with selective affinity for these GABA(A) receptors could promote relaxation of airway smooth muscle. RT-PCR analysis of GABA(A) receptor subunits was performed on RNA isolated by laser capture microdissection from human and guinea pig airway smooth muscle. Membrane potential and chloride-mediated current were measured in response to GABA(A) subunit-selective agonists in cultured human airway smooth muscle cells. Functional relaxation of precontracted guinea pig tracheal rings was assessed in the absence and presence of the α(4)-subunit-selective GABA(A) receptor agonists: gaboxadol, taurine, and a novel 8-methoxy imidazobenzodiazepine (CM-D-45). Only messenger RNA encoding the α(4)- and α(5)-GABA(A) receptor subunits was identified in RNA isolated by laser capture dissection from guinea pig and human airway smooth muscle tissues. Activation of airway smooth muscle GABA(A) receptors with agonists selective for these subunits resulted in appropriate membrane potential changes and chloride currents and promoted relaxation of airway smooth muscle. In conclusion, selective subunit targeting of endogenous airway smooth muscle-specific GABA(A) receptors may represent a novel therapeutic option for patients in severe bronchospasm.     

Norepinephrine reduces ω-conotoxin-sensitive Ca2+ currents in renal afferent neurons in rats

Sympathetic efferent and peptidergic afferent renal nerves likely influence hypertensive and inflammatory kidney disease. Our recent investigation with confocal microscopy revealed that in the kidney sympathetic nerve endings are colocalized with afferent nerve fibers (Ditting T, Tiegs G, Rodionova K, Reeh PW, Neuhuber W, Freisinger W, Veelken R. Am J Physiol Renal Physiol 297: F1427-F1434, 2009; Veelken R, Vogel EM, Hilgers K, Amman K, Hartner A, Sass G, Neuhuber W, Tiegs G. J Am Soc Nephrol 19: 1371-1378, 2008). However, it is not known whether renal afferent nerves are influenced by sympathetic nerve activity. We tested the hypothesis that norepinephrine (NE) influences voltage-gated Ca(2+) channel currents in cultured renal dorsal root ganglion (DRG) neurons, i.e., the first-order neuron of the renal afferent pathway. DRG neurons (T11-L2) retrogradely labeled from the kidney and subsequently cultured, were investigated by whole-cell patch clamp. Voltage-gated calcium channels (VGCC) were investigated by voltage ramps (-100 to +80 mV, 300 ms, every 20 s). NE and appropriate adrenergic receptor antagonists were administered by microperfusion. NE (20 μM) reduced VGCC-mediated currents by 10.4 ± 3.0% (P < 0.01). This reduction was abolished by the α-adrenoreceptor inhibitor phentolamine and the α(2)-adrenoceptor antagonist yohimbine. The β-adrenoreceptor antagonist propranolol and the α(1)-adrenoceptor antagonist prazosin had no effect. The inhibitory effect of NE was abolished when N-type currents were blocked by ω-conotoxin GVIA, but was unaffected by other specific Ca(2+) channel inhibitors (ω-agatoxin IVA; nimodipine). Confocal microscopy revealed sympathetic innervation of DRGs and confirmed colocalization of afferent and efferent fibers within in the kidney. Hence NE released from intrarenal sympathetic nerve endings, or sympathetic fibers within the DRGs, or even circulating catecholamines, may influence the activity of peptidergic afferent nerve fibers through N-type Ca(2+) channels via an α(2)-adrenoceptor-dependent mechanism. However, the exact site and the functional role of this interaction remains to be elucidated.     

GABAA receptors are expressed and facilitate relaxation in airway smooth muscle

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system and exerts its actions via both ionotropic (GABA(A)) channels and metabotropic (GABA(B)) receptors. GABA(A) channels are ubiquitously expressed in neuronal tissues, and in mature neurons modulate an inward chloride current resulting in neuronal inhibition due to membrane hyperpolarization. In airway smooth muscle (ASM) cells, membrane hyperpolarization favors smooth muscle relaxation. Although GABA(A) channels and GABA(B) receptors have been functionally identified on peripheral nerves in the lung, GABA(A) channels have never been identified on ASM itself. We detected the mRNA encoding of the GABA(A) alpha(4)-, alpha(5)-, beta(3)-, delta-, gamma(1-3)-, pi-, and theta-subunits in total RNA isolated from native human and guinea pig ASM and from cultured human ASM cells. Selected immunoblots identified the GABA(A) alpha(4)-, alpha(5)-, beta(3)-, and gamma(2)-subunit proteins in native human and guinea pig ASM and cultured human ASM cells. The GABA(A) beta(3)-subunit protein was immunohistochemically localized to ASM in guinea pig tracheal rings. While muscimol, a specific GABA(A) channel agonist, did not affect the magnitude or the time to peak contractile effect of substance P, it directly concentration dependently relaxed a tachykinin-induced contraction in guinea pig tracheal rings, which was inhibited by the GABA(A)-selective antagonist gabazine. Muscimol also relaxed a contraction induced by an alternative contractile agonist histamine. These results demonstrate that functional GABA(A) channels are expressed on ASM and suggest a novel therapeutic target for the relaxation of ASM in diseases such as asthma and chronic obstructive lung disease.     

Architecture of the myosalpinx of the isthmus in the guinea pig by means of scanning electron microscopy.

The three-dimensional architecture of the isthmic myosalpinx in the guinea pig was investigated by means of a technique that involves stretching the myosalpinx under a dissecting microscope, followed by selective digestion of the interstitial connective tissue with KOH and, finally, observation under a scanning electron microscope. The images obtained in this way prove that the myosalpinx is arranged in single bundles of smooth muscle cells which essentially do not show any specific geometrical course. Furthermore, smooth muscle cell bundles following an oblique course, not referred in the literature prior to this report, were demonstrated. These new three-dimensional microanatomical results call for a reevaluation of the functional role of oviduct motility.     

Role of α1-adrenoceptor subtypes which mediate positive chronotropy in neonatal rat cardiac myocytes

We investigated the involvement of α₁-adrenoceptor subtypes in the positive chronotropic response to norepinephrine (NE) in neonatal rat cardiac myocytes at day 3 of culture. The cardiac myocytes at day 3 of culture exhibited a dose-dependent positive chronotropic response to NE in the presence of propranolol, a β-adrenoceptor antagonist. The positive chronotropic responses to NE were completely antagonized by the α₁-adrenoceptor antagonist prazosin. The NE-induced positive chronotropic response was inhibited 68% by the α_1B-adrenoceptor antagonist, chloroethylclonidine (CEC), but partially (41%) so by the α_1A-adrenoceptor antagonist, WB4101. In the membrane fraction derived from cardiac myocytes at day 3 of culture, pretreatment with CEC decreased the Bmax of the α₁-adrenoceptor to 22% of the control value. The NE-induced positive chronotropic response was inhibited 62 and 77% by the voltage-gated Ca²⁺ channel blocker such as nifedipine and verapamil, respectively. These findings indicate (1) that cultured neonatal rat cardiac myocytes possess both α₁-adrenoceptor subtypes, i.e., α_1A and α_1B, (2) that the predominant α₁-adrenoceptor subtypes mediating NE-induced positive chronotropy in neonatal rat cardiac myocytes at day 3 of culture are α_1B-subtypes, and (3) that NE-induced positive chronotropy may be caused via voltage-gated Ca²⁺ channel activation.     

Excitatory and inhibitory actions of norepinephrine on the Ba2+ current through L-type Ca2+ channels of smooth muscle cells of guinea-pig vas deferens

The effect of norepinephrine (NE) was examined on the whole-cell Ba²⁺ current through L-type Ca²⁺ channels of freshly isolated smooth muscle cells of guinea-pig vas deferens. The magnitude of maximum Ba²⁺ current [IBa(max)] varied in different cells, although the capacitance of the cell membrane was similar (∼50 pF). Application of dbcAMP augmented IBa(max) by 37%, which was canceled by Rp-cAMPs, while PMA decreased the current by 32%, which was canceled by staurosporine. NE increased IBa(max) of the cells which originally showed relatively small IBa(max), and decreased the current of the cells which showed larger IBa(max). In the presence of phentolamine, NE increased IBa(max), and this effect was remarkable in cells showed smaller IBa(max). In the presence of propranolol, NE decreased IBa(max). The excitatory β-adrenoceptor activation was canceled by Rp-cAMPs, and the inhibitory α-adrenoceptor effect was canceled by staurosporine. It is suggested that NE shows dual (excitatory and inhibitory) actions on the L-type Ca²⁺ channels of smooth muscle of guinea-pig vas deferens. The excitatory β-adrenoceptor action mediated through cAMP/PKA is predominant in cells with lower density of the Ca²⁺ channels, while inhibitory α-adrenoceptor action mediated through PKC is predominant in cells with higher channel density. © 1996 Wiley-Liss, Inc.     

The alpha adrenoceptors on endothelial cells

Endothelial cells release a powerful factor (endothelium-derived relaxing factor [EDRF]) that relaxes smooth muscle cells in response to some vasodilating agents such as acetylcholine. Contraction curves to norepinephrine (NE) in greyhound, mongrel dog, and pig coronary artery rings were studied in vitro in the presence of propranolol. Removal of endothelium increased the sensitivity and maximum contraction in response to NE. In other experiments pig coronary rings were precontracted with a thromboxane mimetic U 46619 in the presence of propranolol. NE relaxed these arteries only if endothelium was present. Methoxamine was without effect but the relaxation response to NE was antagonized by phentolamine, idazoxan, and yohimbine, which suggests that there are alpha 2 adrenoceptors on endothelial cells that mediate the release of EDRF. Greyhound and mongrel dog large coronary arteries relaxed to NE only if prazosin was present, which suggests that alpha 1-adrenoceptor stimulation on the vascular smooth muscle can override the relaxation response to EDRF. Comparison of NE responses in carotid, mesenteric, renal, and femoral large arteries of the pig, greyhound, and mongrel dog indicate the nonuniformity of distribution of alpha 2 adrenoceptors on endothelium and alpha 1 and alpha 2 adrenoceptors on vascular smooth muscle. The integrity of the endothelium must now be considered in interpreting the vascular responses to alpha-adrenoceptor agonists.     

Electrical behavior of guinea pig tracheal smooth muscle

Intracellular recordings were taken from the smooth muscle of the guinea pig trachea, and the effects of intrinsic nerve stimulation were examined. Approximately 50% of the cells had stable resting membrane potentials of -50 +/- 1 mV. The remaining cells displayed spontaneous oscillations in membrane potential, which were abolished either by blocking voltage-dependent Ca(2+) channels with nifedipine or by depleting intracellular Ca(2+) stores with ryanodine. In quiescent cells, stimulation with a single impulse evoked an excitatory junction potential (EJP). In 30% of these cells, trains of stimuli evoked an EJP that was followed by oscillations in membrane potential. Transmural nerve stimulation caused an increase in the frequency of spontaneous oscillations. All responses were abolished by the muscarinic-receptor antagonist hyoscine (1 microM). In quiescent cells, nifedipine (1 microM) reduced EJPs by 30%, whereas ryanodine (10 microM) reduced EJPs by 93%. These results suggest that both the release of Ca(2+) from intracellular stores and the influx of Ca(2+) through voltage-dependent Ca(2+) channels are important determinants of spontaneous and nerve-evoked electrical activity of guinea pig tracheal smooth muscle.     

Identification and profiling of novel α1A-adrenoceptor-CXC chemokine receptor 2 heteromer

We have provided the first evidence for specific heteromerization between the α(1A)-adrenoceptor (α(1A)AR) and CXC chemokine receptor 2 (CXCR2) in live cells. α(1A)AR and CXCR2 are both expressed in areas such as the stromal smooth muscle layer of the prostate. By utilizing the G protein-coupled receptor (GPCR) heteromer identification technology on the live cell-based bioluminescence resonance energy transfer (BRET) assay platform, our studies in human embryonic kidney 293 cells have identified norepinephrine-dependent β-arrestin recruitment that was in turn dependent upon co-expression of α(1A)AR with CXCR2. These findings have been supported by co-localization observed using confocal microscopy. This norepinephrine-dependent β-arrestin recruitment was inhibited not only by the α(1)AR antagonist Terazosin but also by the CXCR2-specific allosteric inverse agonist SB265610. Furthermore, Labetalol, which is marketed for hypertension as a nonselective β-adrenoceptor antagonist with α(1)AR antagonist properties, was identified as a heteromer-specific-biased agonist exhibiting partial agonism for inositol phosphate production but essentially full agonism for β-arrestin recruitment at the α(1A)AR-CXCR2 heteromer. Finally, bioluminescence resonance energy transfer studies with both receptors tagged suggest that α(1A)AR-CXCR2 heteromerization occurs constitutively and is not modulated by ligand. These findings support the concept of GPCR heteromer complexes exhibiting distinct pharmacology, thereby providing additional mechanisms through which GPCRs can potentially achieve their diverse biological functions. This has important implications for the use and future development of pharmaceuticals targeting these receptors.     

Clonidine-induced coronary vasodilatation in isolated guinea pig heart is not mediated by endothelial alpha2 adrenoceptors.

Functional role of endothelial alpha(2)-adrenoceptor in coronary circulation remains unclear. Clonidine, an agonist of alpha(2)-adrenoceptors, was reported to induce coronary vasodilatation via stimulation of endothelial alpha(2)-adrenoceptors or coronary vasoconstriction involving vascular smooth muscle alpha(2)-adrenoceptors. Moreover, H(2) receptor-dependent responses to clonidine were described. Here, we reassess the contribution of endothelial alpha(2)-adrenoceptor and H(2) receptors to coronary flow and contractility responses induced by clonidine in the isolated guinea pig heart. We found that clonidine (10(-9) - 10(-6) M) produced concentration-dependent coronary vasoconstriction without a significant change in contractility. This response was inhibited by the alpha(1)/alpha(2)-adrenoceptor antagonist - phentolamine (10(-5) M) and the selective alpha(2)-adrenoceptor antagonist yohimbine (10(-6) M), but it was not changed by the selective alpha(1)-adrenoceptor antagonist prazosin (10(-6) M). In the presence of nitric oxide synthase inhibitor, L-NAME (10(-4) M) the clonidine-induced vasoconstriction was potentiated. Clonidine at high concentrations of 10(-5) - 3 x 10(-5) M produced coronary vasodilatation, and an increase in myocardial contractility. These responses were abolished by a selective H(2)-receptor antagonist, ranitidine (10(-5) M), but not by phentolamine (10(-5) M). We conclude that in the isolated guinea pig heart, clonidine-induced vasoconstriction is mediated by activation of smooth muscle alpha(2)-adrenoceptors whereas clonidine-induced coronary vasodilatation is mediated by activation of vascular H(2) histaminergic receptors. Accordingly, endothelial alpha(2)-adrenoceptors does not seem to play a major role in coronary flow response induced by clonidine.     

内脏平滑肌Cajal间质细胞起搏功能(英文)

胃肠道的大部分区域都存在着一种特殊的间质细胞——Cajal间质细胞(interstitial cells of Cajal,ICCs)。尽管在100多年前它们的存在就已被发现,但是直到最近几十年的研究才逐渐揭示了它们的功能。在胃肠道,ICCs被认为是平滑肌自发性节律性电活动,即"基本电节律"(又称"慢波")的起搏细胞,并介导神经至平滑肌的神经信号传递活动。除胃肠道外,ICC样细胞同样存在于其它内脏平滑肌,如泌尿、生殖系统以及血管平滑肌等。本文仅就这些内脏平滑肌ICCs的功能做一简单综述。     

Mechanism of synaptic inhibition by noradrenaline acting at alpha 2-adrenoceptors

The actions of agonists at alpha 2-adrenoceptors were investigated on single cells of the submucous plexus of the guinea pig small intestine. Intracellular recordings were made from neurons in vitro, and noradrenaline and other agonists were applied by adding them to the superfusion solution. The actions of noradrenaline released from terminals of sympathetic nerves was also studied by stimulating the nerves and recording the inhibitory postsynaptic current; this current can be mimicked by brief applications of noradrenaline from a pipette tip positioned within 50 micron of the neuron. The alpha 2-adrenoceptor-bound noradrenaline with an apparent dissociation constant of 15 microM, determined by the method of partial irreversible receptor inactivation: clonidine and 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14304) had dissociation constants of 36 nM and 2.5 microM respectively. Noradrenaline and UK 14304 caused maximal hyperpolarizations, or outward currents; clonidine was a full agonist in only 4 of 35 cells, a partial agonist in 25 cells, and without effect in 4 cells. Clonidine acted as a competitive antagonist of noradrenaline in those cells in which it lacked agonist action; its dissociation equilibrium constant determined by Schild analysis was about 20 nM. The potassium conductance increased by the alpha 2-adrenoceptor agonists, whether they were applied exogenously or released by stimulation of presynaptic nerves, showed marked inward rectification. The neurons showed inward rectification also in the absence of agonist; both types of rectification were eliminated by rubidium (2 mM), barium (3-30 microM) and caesium (2 mM). When the recording electrodes contained the nonhydrolysable derivative of guanosine 5'-triphosphate (GTP), guanosine 5'-O-(3-thiotriphosphate, GTP-gamma-S), the effects of applied alpha 2-adrenoceptor agonists did not reverse when they were washed from the tissue, implying that GTP hydrolysis is necessary for the termination of agonist action. Pretreatment with pertussis toxin abolished the inhibitory synaptic potential (IPSP) and agonist-induced hyperpolarizations. Phorbol 12,13-dibutyrate, forskolin, cholera toxin and sodium fluoride did not affect the responses to alpha 2-adrenoceptor agonists. The synaptic hyperpolarization resulting from sympathetic nerve stimulation, or the hyperpolarization evoked by a brief (3-5 ms) application of noradrenaline, began after a latency of about 30 and 60 ms respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Bradykinin receptors: functional similarities in guinea pig gut muscle and mucosa

It is well established that bradykinin can stimulate mucosal electrolyte transport. However, the receptor type which mediates this effect has not been fully characterized. Recent studies have suggested that bradykinin and related kinins may act at two types of receptors designated as B1 and B2. We have determined the effect of bradykinin on electrolyte secretion across guinea pig ileal mucosa and longitudinal muscle in vitro in the presence and absence of D-Phe7-bradykinin (B2 antagonist) and des-Arg9-(Leu8)-bradykinin (B1 antagonist). The B2 antagonist (less than 100 microM) did not affect resting muscle tension or basal electrolyte transport but at 6-30 microM it caused a parallel rightward shift in the concentration-response curves to bradykinin in the mucosa (Ki = 4 microM) and muscle (Ki = 6 microM). Changes in electrolyte transport and muscle contractility evoked by bethanechol and substance P were not affected by the B2 antagonist (30 microM) in either the muscle or the mucosa. Moreover, changes in electrolyte transport and muscle contractility produced by bradykinin were not altered by the B1 antagonist (30 microM). Finally, the B1 agonist des-Arg9-bradykinin (10 nM-1 microM) was not active in either preparation. These data suggest that under normal conditions, ileal secretion and smooth muscle contractility in the guinea pig are regulated by B2-type bradykinin receptors.     

Direct contractile effect of motilin on isolated smooth muscle cells of guinea pig small intestine.

We examined the direct effect of motilin on longitudinal and circular smooth muscle cells isolated from the guinea pig small intestine. In addition, the effects of 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxy-benzoate hydrochloride (TMB-8, an inhibitor of intracellular Ca(2+)-release), verapamil (a voltage-dependent Ca(2+)-channel blocker), and removal of extracellular Ca2+ were investigated to evaluate the role of intracellular Ca2+ stores and extracellular Ca2+ on the muscle contraction induced by motilin. The effects of atropine (a muscarinic receptor antagonist), spantide (a substance P receptor antagonist) and loxiglumide (a CCK-receptor antagonist) were also examined to determine whether the motilin-induced contraction was independent of those receptors. Motilin induced a contraction of the longitudinal and circular smooth muscle cells in a dose-dependent manner with the maximal effect attained after 30 seconds of incubation. The ED50 values were 0.3 nM and 0.05 nM, respectively. TMB-8 suppressed completely the motilin-induced contraction of both types of smooth muscle cells. Verapamil had only a slight suppressive effect. Removal of extracellular Ca2+ did not have any significant influence on motilin-induced contraction. The contractile response to motilin was not affected by atropine, spantide or loxiglumide. Our findings showed that:1) motilin has a direct contractile effect on both longitudinal and circular smooth muscle cells; 2) this contractile effect is not evoked via muscarinic, substance P or CCK receptors, and 3) the intracellular release of Ca2+ plays an important role in the contractile response to motilin on both types of smooth muscle cells.     

Regulation of catecholamine release in human adrenal chromaffin cells by β-adrenoceptors

The adrenal gland plays a fundamental role in the response to a variety of stress situations. After a stress condition, adrenal medullary chromaffin cells release, by exocytosis, high quantities of catecholamine (epinephrine, EP; norepinephrine, NE), especially EP. Once in the blood stream, catecholamines reach different target organs, and induce their biological actions through the activation of different adrenoceptors. Adrenal gland cells may also be activated by catecholamines, through hormonal, paracrine and/or autocrine system. The presence of functional adrenoceptors on human adrenal medulla and their involvement on catecholamines secretion was not previously evaluated. In the present study we investigated the role of β(1)-, β(2)- and β(3)-adrenoceptors on catecholamine release from human adrenal chromaffin cells in culture. We observed that the β-adrenoceptor agonist (isoproterenol) and β(2)-adrenoceptor agonist (salbutamol) stimulated catecholamine (NE and EP) release from human adrenal chromaffin cells. Furthermore, the β(2)-adrenoceptor antagonist (ICI 118,551; 100 nM) and β(3)-adrenoceptor antagonist (SR 59230A; 100 nM) inhibited the catecholamine release stimulated by isoproterenol and nicotine in chromaffin cells. The β(1)-adrenoceptor antagonist (atenolol; 100 nM) did not change the isoproterenol- neither the nicotine-evoked catecholamine release from human adrenal chromaffin cells. Moreover, our results show that the protein kinase A (PKA), protein kinase C (PKC), mitogen-activated protein kinase (MAPK) and phospholipase C (PLC) are intracellular mechanisms involved in the catecholamine release evoked by salbutamol. In conclusion, our data suggest that the activation of β(2)- and β(3)-adrenoceptors modulate the basal and evoked catecholamine release, NE and EP, via an autocrine positive feedback loop in human adrenal chromaffin cells.     

Mathematical description of regenerative potentials recorded from circular smooth muscle of guinea pig antrum

Regenerative potentials evoked by intracellular current injection in single bundles of circular smooth muscle taken from guinea pig antrum have the characteristics of the secondary regenerative component of the slow wave occurring in the same muscle layer. Such regenerative depolarizations might result from a mechanism that responds to membrane polarization with a delayed increase in the rate of production of unitary potentials detected in this tissue. To test this possibility, a two-stage reaction leading to the formation of an intracellular messenger was proposed. The first forward reaction was voltage-dependent, in the manner described by the Hodgkin-Huxley transient Na conductance formalism, allowing simulation of anode break excitation, stimulus threshold strength-duration characteristics, and refractory behavior. A conventional dose-effect relationship was proposed to describe the dependence of the mean rate of discharge of unitary potentials on messenger concentration. Unitary potentials were modeled as unitary membrane conductance modulations with an empirically derived amplitude distribution and Poisson-distributed intervals. The model reproduces a range of spontaneous and evoked membrane potential changes characteristic of antral circular muscle bundles.     

Neuropeptide Y-like immunoreactivity in intramural ganglia of the newborn guinea pig urinary bladder

Immunoreactive neuropeptide Y (NPY) was demonstrated in neuronal elements in the urinary bladder wall of the newborn guinea pig. Numerous intramural ganglia were found lying among the smooth muscle bundles and in the submucosa, and NPY-like immunoreactive nerve cell bodies were demonstrated within all of these ganglia. Nerve fibres containing NPY were also richly distributed in the detrusor muscle, submucosa and around blood vessels. In dissociated cell cultures from newborn guinea pig detrusor muscle, a subpopulation (70-85%) of both mononucleate and binucleate intramural neurones was shown to contain NPY-like immunoreactivity. A low percentage (1-6%) of the intramural bladder neurones contained dopamine-beta-hydroxylase. In conclusion, while some NPY-containing nerve fibres in the wall of the bladder are of sympathetic origin, especially those supplying blood vessels, the results of this present study establish that many of these NPY-containing nerve fibres originate from non-adrenergic cell bodies within the intramural bladder ganglia.