Spontaneous Mechanical Activity Research Articles

Regulation of the spontaneous contractile activity of the portal vein by the sarcoplasmic reticulum: evidence from the phospholamban gene-ablated mouse

The rapid contraction/relaxation cycles of phasic smooth muscles necessitates intracellular calcium cycling at a more rapid rate than that of tonic smooth muscles. Recent studies suggest that sarcoplasmic reticulum calcium handling is an important determinant of portal vein phasic contractions. We evaluated the importance and role of phospholamban, a protein which inhibits the sarcoplasmic reticulum (SR) calcium ATPase (SERCA), in regulating the contractility of the phasic mouse portal vein. PLB gene ablation significantly reduced the basal frequency of spontaneous mechanical activity and increased force development of the portal vein. Cyclopiazonic acid (CPA), an inhibitor of SERCA, did not significantly affect the spontaneous activity of the wild-type (WT) portal vein. CPA (1 μM) eliminated the differences in frequency and force between the PLB-KO and WT, localizing the effects to the SR. The PLB-KO portal vein had a lower resting membrane potential than WT controls. There were no significant differences between WT and KO responses to charybdotoxin (250 nM), indicating that calcium-activated potassium channels do not contribute to altered KO portal vein contractility. While contractile sensitivity to acetylcholine was not different between WT and PLB-KO portal veins, force generated in response to a given concentration of acetylcholine was significantly greater in the PLB-KO portal vein, both in the absence and presence of CPA. Our results confirm that SR activity can play a major role in modulating the frequency of the spontaneous mechanical activity of portal veins and removal of PLB inhibition of the SR calcium ATPase has significant effects on the spontaneous activity of the portal vein.

A role for chromogranin A (4–16), a vasostatin-derived peptide, on human colonic motility. An in vitro study

The hypothesis that CgA-derived peptides may be involved in mechanisms modulating motility was tested. Human colonic smooth muscles were studied using an organ bath technique. Acetic acid (AA) effects were characterized on spontaneous mechanical activities (SMA) and on responses to transmural nerve stimulation (NS). AA induced a significant decrease in tone and abolished SMA; this effect was insensitive to either TTX or l-NAME/apamin. The AA-induced inhibitory effects were significantly reduced in the presence of CgA4–16. This effect was insensitive to TTX or l-NAME/apamin. Furthermore, AA-induced effects were blocked in the presence of BAYK8644 and CgA4–16 together. The inhibitory effect of nifedipine was delayed in the presence of CgA4–16. NS induced a triphasic response. Only the excitatory components were reduced in the presence of AA. This effect was dose-related and remained unchanged in the presence of CgA4–16 alone, but was blocked in the presence of simultaneous administration of CgA4–16 and l-NAME/apamin. AA application induced inhibition of human colon motility in vitro. This effect may be mediated through an action on L-type calcium channels. CgA4–16 may display a protective role, which prevents the inhibition of motility due to AA to occur, by acting on both smooth muscle and afferent terminals.

Investigation into gastrointestinal pacemaker mechanism using cultured cell cluster preparation

Gastrointestinal tract motility is driven by pacemaker depolarization referred to slow waves. In order to investigate mechanisms underlying the spontaneous rhythmicity, we have developed a cell cluster preparation. Cell clusters were enzymatically isolated from the muscle layers of mouse small intestine and cultured for several days. They include smooth muscle, enteric neurons and c-Kit-immunopositive cells (interstitial cells of Cajal: ICC), and preserve spontaneous mechanical and electrical activities. A characteristic feature of the pacemaker potential is resistance to dihydropyridine (DHP) Ca(2+) antagonist. In the presence of nifedipine, a DHP Ca(2+) antagonist, spontaneous intracellular Ca(2+) ([Ca(2+)](i)) oscillation was recorded from c-Kit-immunopositive cells in the cell cluster preparation. The [Ca(2+)](i) oscillation seen in ICC was terminated by applications of drugs affecting ryanodine receptors as well as those for InsP(3) receptors and TRP family channels. It is considered that these intracellular Ca(2+) release channels and the Ca(2+) influx pathway from the extracellular space cooperate to produce pacemaker activity in the gastrointestinal tract.

Nitric oxide induces muscular relaxation via cyclic GMP-dependent and -independent mechanisms in the longitudinal muscle of the mouse duodenum

The aim of this study was to investigate, in mouse duodenum, the role of nitric oxide (NO) in the relaxation of longitudinal muscle evoked by nerve activation and the coupled action mechanism. Electrical field stimulation (EFS; 0.5 ms, 10-s train duration, supramaximal voltage, at various frequencies) under nonadrenergic noncholinergic conditions evoked muscular relaxation occasionally followed, at the higher stimulus frequencies, by rebound contractions. Inhibition of the synthesis of NO by N ω -nitro- l-arginine methyl ester ( l-NAME; 100 μM) virtually abolished the evoked relaxation. The relaxation was reduced also by apamin (0.1 μM) and by 1H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one (ODQ; 1 μM), a guanylyl cyclase inhibitor. The coadministration of apamin and ODQ produced additive effects on the responses to EFS. Sodium nitroprusside (0.1–100 μM) produced a concentration-dependent reduction of the phasic spontaneous activity and at the highest dose used suppressed phasic activity and induced muscular relaxation. These effects were tetrodotoxin and l-NAME resistant and were antagonized both by apamin and by ODQ. 8-Bromoguanosine 3 ′,5 ′-cyclic monophosphate (0.1–100 μM) reduced in a concentration-dependent manner the spontaneous mechanical activity and at 100 μM suppressed the phasic activity and induced muscular relaxation, not antagonized by apamin. This study indicates that NO is the primary transmitter released by inhibitory nerves supplying the longitudinal muscle of mouse duodenum and that guanylate cyclase stimulation and opening of Ca 2+-dependent K + channels are independent mechanisms working in parallel to mediate NO action.

Evidence that action potential generation is not the exclusive determinant to trigger spontaneous myogenic contraction of guinea-pig urinary bladder smooth muscle.

Urinary bladder smooth muscle (UBSM) exhibits spontaneous contraction. This spontaneous mechanical activity is myogenic and can be closely related to the UBSM cell action potential to facilitate Ca2+ influx through voltage-gated Ca2+ channels. In the present study, to know whether this membrane electrical event is the exclusive mechanism to trigger spontaneous smooth muscle contraction, we compared the inhibitory effects of Ca2+ channel blockers on the spontaneous action potential and mechanical activity in the isolated guinea-pig UBSM. Both action potential and rhythmic contraction were generated spontaneously in the presence of atropine (1 microM), phentolamine (1 microM), propranolol (1 microM), suramin (10 microM) and tetrodotoxin (1 microM), which suggest that both phenomena were myogenic in origin. Nisoldipine (100 nM) and diltiazem (10 microM) completely eliminated the generation of action potential whereas its frequency was dramatically increased by a dihydropyridine Ca2+ agonist, BayK 8644 (1 microM). In contrast to disappearance of action potential in the presence of Ca2+ channel blockers, spontaneous contraction of UBSM was inhibited only partly by nisoldipine or diltiazem and most of the mechanical components persisted in these channel blockers. These results indicate that spontaneous action potential in UBSM cell is generated through the activation of L-type voltage-gated Ca2+ channels. The subsequent elevation of intracellular Ca2+ concentrations during a burst of action potentials can be partly responsible for the induction of UBSM mechanical activity. In addition, the present study provides evidence that UBSM spontaneous mechanical activity is also attributable to the mechanism(s) other than the generation of Ca2+ spike.

Control of non-adrenergic non-cholinergic reflex motor responses in circular muscle of guinea-pig small intestine by Met-enkephalin.

1 A triple organ bath method allowing the synchronous recording of the motor activity of the circular muscle layer belonging to the oral and anal segments of guinea-pig small intestine adjacent to an electrically stimulated middle segment was developed to study the ascending and descending reflex motor responses. 2 Electrical field stimulation (0.8 ms, 40 V, 5 Hz, 10 s) applied to the middle part of the segments elicited tetrodotoxin (1 microm)-sensitive ascending and descending contractile responses of the nonstimulated parts, oral and anal, respectively. The ascending contraction was more pronounced as compared with the descending contraction. 3 In the presence of phentolamine (5 microm), propranolol (5 microm) and atropine (3 microm) a significant decrease in the amplitude of the ascending contraction was seen and a descending relaxation, instead of a contraction was observed. 4 Met-enkephalin applied at a single concentration (0.1 microm) or cumulatively (0.001-1 microm) inhibited both non-adrenergic non-cholinergic (NANC) descending relaxation and ascending contraction with similar efficacy but different potency, IC50 being 5.9 +/- 0.3 and 39.0 +/- 4 nm, respectively. Naloxone (0.5 microm) prevented the effects of Met-enkephalin. 5 L-NNA (0.5 mm), an inhibitor of nitric oxide synthesis, increased the ascending contraction and strongly reduced but not abolished the descending relaxation. l-Arginine (0.5 mm) restored the motor responses to the initial level in l-NNA-pretreated preparations, d-Arginine (0.5 nm) had no effects. 6 Met-enkephalin (0.1 microm) depressed the l-NNA-dependent increase of the ascending contraction and failed to change the l-NNA-resistant part of the descending relaxation. 7 Met-enkephalin did not alter spontaneous NANC mechanical activity. SNP (1 or 10 microm), an exogenous donor of nitric oxide, caused a concentration-dependent relaxation. The effects of SNP persisted in Met-enkephalin (0.1 microm)-pretreated preparations. 8 NANC reflex ascending contraction and descending relaxation were synchronously induced by a local nerve stimulation indicating a functional coactivation of NANC orally projected excitatory and anally directed inhibitory pathways. Acting prejunctionally, Met-enkephalin provided a negative controlling mechanism inhibiting both ascending and descending, mainly nitric oxide mediated, reflex responses. A higher sensitivity of the descending relaxation to Met-enkephalin was observed suggesting an essential role of opioid(s) in reducing the efficacy of descending motor activity.

Effects of GABA on circular smooth muscle spontaneous activities of rat distal colon

GABAergic regulation of intestinal motility through the modulation of non-adrenergic non-cholinergic (NANC) neurons remains poorly understood especially in rat colon where very few studies have been undertaken. Thperefore, the effects of GABA on circular preparations of rat distal colon were investigated using classical organ bath chambers to record spontaneous mechanical activities (SMA). SMA was characterized by the occurrence of rhythmic phasic contractions (type-I) or by spontaneously occurring large contractions superimposed on small rhythmic contractions (type-II). In the presence of atropine and guanethidine (NANC conditions), these large contractions were inhibited by bicuculline, a GABA A-receptor antagonist as well as by TTX, L-NAME and apamin together, or L 732-138, a NK1-receptor antagonist. In NANC conditions, GABA induced a transient monophasic relaxation or a biphasic effect characterized by a relaxation followed by a tonic contraction in both type-I and -II preparations. Both the inhibitory and excitatory effects of GABA were blocked by TTX and L-NAME + apamin; the GABA-induced contraction was also sensitive to L 732-138. The responses to GABA were mimicked by the GABA A-receptor agonist, muscimol, whereas baclofen and CACA, respectively GABA B and GABA C-receptors agonists showed no effect. These results demonstrated that only GABA A-receptors seem to be involved in the regulation of SMA in rat distal colon in NANC conditions. Release of NANC inhibitory transmitter (NO and probably ATP) and NANC excitatory transmitter (maybe substance P) might be involved.

BK channels play an important role as a negative feedback mechanism in the regulation of spontaneous rhythmic contraction of urinary bladder smooth muscles

Smooth muscles of urinary bladder wall exhibit spontaneous rhythmic contraction which is myogenic in origin. Although the precise mechanism responsible for the generation of this mechanical activity remains to be established, it can be related closely to the action potential (AP) in urinary bladder smooth muscle (UBSM) cell, and may be the fundamental constituent to determine urinary bladder physiological functions to store and micturate urine. In the present study, possible roles of voltage-dependent and Ca(2+)-sensitive K+ (BK) channels, highly expressed in UBSM cells, were examined in the regulation of spontaneous UBSM contraction with reference to the generation of AP. Iberiotoxin (IbTx), a selective BK channel blocker, strongly increased mechanical activity and AP generation in guinea-pig UBSM. In contrast, BK channel openers (NS-1619, niflumic acid; estradiol, tamoxifen: BK channel alpha- and beta-subunit activators, respectively) significantly diminished AP generation and spontaneous mechanical activity. The present study indicates that BK channels play the primary role as a negative feedback element to limit extracellular Ca2+ influx through affecting AP configurations in the generation of UBSM contraction. BK channel openers including beta-subunit activators may be a potentially useful therapeutic remedy for the treatment of urinary bladder dysfunctions such as frequent urination.

Involvement of tachykinin NK2 receptors in the modulation of spontaneous motility in rat proximal colon.

The role of endogenous tachykinins and the mechanisms whereby they act on NK2 receptors, modulating spontaneous motility, were investigated in rat isolated proximal colon. The mechanical activity was detected as changes in intraluminal pressure. The NK2 receptor antagonist, MEN 10627, produced a concentration-dependent reduction of the contraction amplitude. [beta-Ala8]-neurokinin A(4-10), an NK2 receptor agonist, and [Sar9, Met(O2)11]-Substance P ([Sar9, Met(O2)11]-SP), an NK1 receptor agonist, induced a concentration-dependent contractile response, characterized by an increase in basal tone with superimposed phasic contractions. MEN 10627 antagonized the response to [beta-Ala8]-neurokinin A(4-10), without affecting that to [Sar9, Met(O2)11]-SP. Tetrodotoxin (TTX), hexamethonium and Nomega-nitro-L-arginine methyl ester (L-NAME) significantly reduced the response to MEN 10627. The NK3 receptor agonist, senktide, was able to activate the nitrergic inhibitory pathway, as it induced a TTX-and L-NAME-sensitive inhibitory effect. [beta-Ala8]-neurokinin A(4-10) was able to antagonize the inhibitory response to senktide. These findings suggest that tachykinins acting on NK2 receptors play a role in the modulation of the spontaneous mechanical activity. The mechanism of this action would be, in part, acting directly on the smooth muscle cells, and, in part neurogenic, sustained by nicotinic inputs, and possibly due to inhibition of NO tonic release.

Electrical and mechanical effects of vasoactive intestinal peptide and pituitary adenylate cyclase-activating peptide in the rat colon involve different mechanisms

This work aimed to study the effects of pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) on the mechanical and electrical activity of the circular muscle of the rat colon and the mechanisms involved in such effects. Spontaneous mechanical activity was studied in vitro in an organ bath and the membrane potential was recorded using the microelectrode technique. Both VIP and PACAP (0.1 μM) caused an immediate, sustained and tetrodotoxin (1 μM)-resistant inhibition of the cyclic spontaneous mechanical activity and hyperpolarization. The small-conductance Ca 2+-activated K + channel blocker, apamin (1 μM), did not change the VIP- and PACAP-induced relaxation but reduced the hyperpolarization induced by PACAP whereas it did not change that induced by VIP. In contrast, the purinoceptor antagonist, suramin (100 μM), blocked the hyperpolarization caused by PACAP and VIP but failed to change their mechanical inhibitory effects. Moreover, the putative PACAP and VIP receptor antagonists, PACAP-(6–38) and VIP-(10–28), respectively, both 3 μM, failed to change the effects of either peptide and modified neither the inhibitory junction potential nor the relaxation induced by electrical-field stimulation. Thus, these results suggest that the mechanisms mediating relaxation are not strictly coupled to the mechanisms mediating hyperpolarization. This could be due to activation of two distinct mechanisms of action after agonist receptor interaction.

Lack of carbachol response indicates the absence of cholinergic receptors in sacs associated with undescended testis

Background/Purpose: The mechanism of testicular descent remains controversial. The processus vaginalis (PV) contains smooth muscle and should have contractile activity that may contribute to descent. This study was designed to evaluate the smooth muscle of PVs associated with incomplete obliteration for spontaneous activities and responses to various stimuli, to determine if differences exist according to sex, diagnostic source, or location of the testis. Materials: Peritoneal samples (n = 4); sacs from girls (n = 8) and boys with inguinal hernia (n = 12); and sacs from boys with hydrocele (n = 3), hydrocele of the cord (n = 2), or undescended testis (n = 7) were used for the current study. Tissues were attached to the isometric force displacement transducer in an organ bath containing mammalian Ringer's solution at 37°C. Spontaneous mechanical activity and contractile responses of tissues to the electrical field stimulation, phenylephrine, carbachol, and serotonin were recorded. The values obtained from boys and girls with inguinal hernia and from boys with either undescended or descended testis were compared through Fisher's Exact test. Results: There were no statistically significant differences in patient age between groups. Among the parameters studied, only the carbachol response of the sacs associated with undescended testis showed a significant difference compared with the others ( P = .001). None of the sacs associated with undescended testis responded to carbachol, whereas all of the sacs from boys and girls with inguinal hernia responded to carbachol. Conclusions: Lack of carbachol response suggests the absence of cholinergic receptors within the sacs associated with undescended testis. The lack of cholinergic receptors may play a role in the failure of the process of testicular descent by hindering either PV elongation into the scrotum or a possible propulsive activity of the PV on the testis.

Tonic inhibitory action by nitric oxide on spontaneous mechanical activity in rat proximal colon: involvement of cyclic GMP and apamin-sensitive K+ channels.

1. The cellular mechanisms by which endogenous nitric oxide (NO) modulates spontaneous motility were investigated in rat isolated proximal colon. The mechanical activity was detected as changes in intraluminal pressure. 2. Apamin (1-100 nM) produced a concentration-dependent increase in the amplitude of the spontaneous pressure waves. The maximal contractile effect was of the same degree as that produced by Nomega-nitro-L-arginine methyl ester (L-NAME) (100 microM) and the joint application of apamin plus L-NAME had no additive effects. Apamin (0.1 microM) reduced the inhibitory effects (i.e. reduction in the amplitude of the pressure waves) induced by sodium nitroprusside (SNP) (1 nM - 10 microM) or 8-Br-cyclic GMP (1-100 microM). 3. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (0.1-5 microM), inhibitor of NO-stimulated guanylate cyclase, produced a concentration-dependent increase of the spontaneous contractions. ODQ (1 microM) in the presence of apamin (0.1 microM) did not produce any further increase in the contraction amplitude, whereas after L-NAME (100 microM) it decreased the spontaneous contractions. ODQ (1 microM) reduced the SNP inhibitory effects. 4. Zaprinast (1-50 microM), inhibitor of cyclic GMP phosphodiesterase, produced a concentration-dependent decrease of the spontaneous contractions. The effects of zaprinast were significantly reduced in the presence of apamin (0.1 microM) or L-NAME (100 microM). 5. These results suggest that small conductance Ca2+-dependent K+ channels and cyclic GMP are involved in the modulation of the spontaneous contractile activity in rat proximal colon. Cyclic GMP production system and opening of apamin-sensitive K+ channels appear to work sequentially in transducing an endogenous NO signal.

Modulation by nitric oxide of spontaneous mechanical activity in rat proximal colon.

1. In order to examine the role of nitric oxide (NO) in the tonic neural inhibition in rat proximal colon, the effects of N(omega)-nitro-L-arginine methyl ester (L-NAME) were studied on the spontaneous contractions of circular muscle (monitored as intraluminal pressure changes) and of longitudinal muscle (detected as isometric tension changes). 2. L-NAME (3 x 10(-6)-3 x 10(-4) M) caused a concentration-dependent increase in the amplitude of circular contractions, without affecting those of longitudinal muscle. This effect was prevented by L-arginine (1-5 x 10(-3) M), but not D-arginine. 3. In the presence of tetrodotoxin (10(-6) M), which per se induced increase of the pressure waves, L-NAME (10(-4) M) caused no further effects on the amplitude of the spontaneous contractions. 4. The response to L-NAME (10(-4) M) was unaffected by atropine (10(-6) M), guanethidine (10(-6) M), hexamethonium (up to 3 x 10(-4) M) or alpha-chymotrypsin (up to 5 U ml(-1)). 5. NK2 receptor antagonists, SR 48968 (3 x 10(-6) M) or MEN 10627 (10(-6) M), produced a reduction of the amplitude of the pressure waves but failed to affect the contractile response to L-NAME (10(-4) M). 6. These findings suggest that tonic production of NO from inhibitory neurones influences the degree of contractions of circular muscle. An involvement of an inhibitory peptide as well as disinhibition of cholinergic or NK2-tachykinergic excitatory neurotransmission in the mechanism of NO action can be ruled out.

Consequences of metabolic inhibition in smooth muscle isolated from guinea-pig stomach.

1. In smooth muscle isolated from the guinea-pig stomach, cyanide (CN) and iodoacetic acid (IAA) were applied to block oxidative phosphorylation and glycolysis, respectively. Effects of IAA on generation of spontaneous mechanical and electrical activities were systematically investigated by comparing those of CN. Spontaneous activity ceased in 10-20 min during applications of 1 mM IAA. On the other hand, application of 1 mM CN also reduced the spontaneous activity, but never terminated it. In the presence of CN the negativity of the resting membrane potential was slightly reduced. 2. When spontaneous activity ceased with IAA, the resting membrane potential was not significantly affected. Also, before ceasing, the amplitude and duration of the spontaneous electrical activity were significantly reduced. The amplitude of the electrotonic potential was, however, not changed by IAA. Further, glibenclamide did not prevent the effects of IAA. These results suggest that, unlike cardiac muscle, activation of metabolism-dependent K+ channels in stomach smooth muscle does not seem to play a major role in reducing and terminating spontaneous activity during metabolic inhibition. 3. Carbachol-induced contraction transiently increased, and subsequently decreased gradually during application of IAA. 4. After 50 min application of IAA, when there was no spontaneous activity, the concentrations of phosphocreatine (PCr) and ATP measured with 31P nuclear magnetic resonance decreased to 60 and 80% of the control, respectively, while inorganic phosphate (Pi) concentration paradoxically fell to below detectable levels. During subsequent prolonged application of IAA, high-energy phosphates steadily decreased. On the other hand, after 50 min CN application, [PCr] and [ATP] decreased to approximately 30 and 80% of the control, respectively, while [Pi] increased by 2.6-fold. 5. In the presence of either CN or IAA, spontaneous mechanical and electrical activities were reduced or eliminated, although amounts of high-energy phosphates sufficient to contract smooth muscle remained. It can be postulated that some mechanism(s) related to energy metabolism, but not including ATP-sensitive K+ channels, plays an important role in generating spontaneous activity in guinea-pig stomach smooth muscle. During metabolic inhibition the energy metabolism-dependent mechanism(s) would preserve high-energy phosphates, and consequently cell viability, by stopping spontaneous activity.

Influence of nitric oxide and vasoactive intestinal peptide on the spontaneous and triggered electrical and mechanical activities of the canine ileum

Modulation of canine ileal pacemaker activity by nitric oxide (NO) or vasoactive intestinal peptide (VIP) was studied during recording of the intracellular electrical and mechanical activity from the entire muscularis externa and from an isolated circular muscle preparation both cut in the long axis of the circular muscle. In the whole-thickness preparation with cholinergic and adrenergic nerve function blocked, the inhibitory junction potentials (IJPs) recorded near the myenteric plexus (MyP) or deep muscular plexus (DMP) were abolished by omega-conotoxin GVIA (omega-CTX, 10(-7) to 3 x 10(-7) M), tetrodotoxin (TTX, 1 microM), or the NO synthase (NOS) inhibitor N omega-nitro-L-arginine (L-NNA at 50 microM). IJPs from electrical field stimulation triggered slow waves (TSWs); after TTX or omega-CTX, TSWs still occurred, advanced in time and increased in amplitude after TTX. Addition of L-NNA advanced the onset of the TSWs after omega-CTX. TTX, L-NNA, or omega-CTX left the resting membrane potentials, the characteristics of spontaneous slow waves, or TSWs evoked by a long stimulating pulse unchanged. L-NNA at 100 microM enhanced the amplitude but not the frequency of spontaneous slow waves. TTX and NOS blockers all increased circular muscle contractions associated with the spontaneous slow waves and TSWs. In isolated circular muscle preparations, the NOS inhibitors N omega-nitro-L-arginine methyl ester (L-NAME at 300 microM) or L-NNA at 100 microM abolished the IJPs and increased the regularity and amplitude of spontaneous slow waves and associated contractions, but TSWs could not be evoked before or after NOS inhibition. The NO donor 3-morpholinosydnonimine hydrochloride (SIN-1) at 200 microM caused hyperpolarizations (10-15 mV) similar to the IJP mediator, attenuated the IJPs, and abolished mechanical activities. SIN-1 increased the slow wave frequency but decreased the amplitude and duration of spontaneous slow waves and TSWs. VIP (10(-6) M) decreased contraction and slow wave amplitude and prolonged IJP duration without affecting membrane potential or slow wave frequency. We conclude that spontaneous slow waves and TSWs originate independently of neural activity. Pacemaking regions possess inhibitory neural inputs that release NO to mediate IJPs and relaxation and influence the delay before a TSW. NO (not VIP) release from nerves inhibits initiation of spontaneous slow waves or TSWs near the MyP, and spontaneous NO release modulates pacemaking activity from the DMP.

Pharmacological characterization of the inwardly-rectifying current in the smooth muscle cells of the rat bladder.

1. In freshly-isolated single cells of the rat bladder detrusor, outwardly-rectifying and inwardly-rectifying membrane currents were identified by the whole-cell voltage-clamp technique. 2. The inwardly-rectifying current (IIR) exhibited features of a cation current permeable to both K+ and Na+ but it was unaffected by changes in extracellular Ca2+. It had an activation threshold close to -60 mV and an estimated reversal potential of -29 mV. 3. IIR activated slowly with a voltage-sensitive time-constant of 69 ms at -140 mV and 209 ms at -100 mV but did not exhibit time-dependent inactivation. 4. IIR was unaffected by tetraethylammonium (up to 20 mM) but it was reduced by extracellular Ba2+ (1 mM) and by extracellular Cs+ (1 mM). 5. IIR was reduced by terikalant (100 microM) and markedly inhibited by ciclazindol (100 microM) although at these concentrations, both agents also reduced outward currents. 6. IIR was inhibited by ZD7288 (10-100 microM) in a concentration-dependent manner. At concentrations up to 30 microM, ZD7288 did not reduce the magnitude of outward currents but these were inhibited by 100 microM ZD7288. 7. In strips of bladder detrusor, spontaneous mechanical activity was increased by ZD7288 (0.3-100 microM) and by ciclazindol (0.3-100 microM) but was unaffected by glibenclamide (1-10 microM). 8. It is concluded that IIR closely resembles the hyperpolarization-activated current Ih, previously described in the smooth muscle of rabbit jejunum and in a variety of other cell types. This current may play an important role in modulating detrusor excitability but this could not be confirmed using the inhibitors ZD7288 and ciclazindol.

Modulation of membrane currents and mechanical activity by niflumic acid in rat vascular smooth muscle

The effects of niflumic acid on whole-cell membrane currents and mechanical activity were examined in the rat portal vein. In freshly dispersed portal vein cells clamped at −60 mV in caesium (Cs +)-containing solutions, niflumic acid (1–100 μM) inhibited calcium (Ca 2+)-activated chloride currents ( I Cl(Ca)) induced by caffeine (10 mM) and by noradrenaline (10 μM). In a potassium (K +)-containing solution and at a holding potential of −10 mV, niflumic acid (10–100 μM) induced an outward K + current ( I K(ATP)) which was sensitive to glibenclamide (10–30 μM). At concentrations < 30 μM and at a holding potential of −2 mV, niflumic acid had no effect on the magnitude of the caffeine- or noradrenaline-stimulated current ( I BK(Ca)) carried by the large conductance, Ca 2+-sensitive K + channel (BK Ca). However, at a concentration of 100 μM, niflumic acid significantly inhibited I BK(Ca) evoked by caffeine (10 mM) but not by NS1619 (1-(2′-hydroxy-5′-trifluoromethylphenyl)-5-trifluorommethyl-2(3 H)benzimidazolone; 20 μM). In Cs +-containing solutions, niflumic acid (10–100 μM) did not inhibit voltage-sensitive Ca 2+ currents. In intact portal veins, niflumic acid (1–300 μM) inhibited spontaneous mechanical activity, an action which was partially antagonised by glibenclamide (1–10 μM), and contractions produced by noradrenaline (10 μM), an effect which was glibenclamide-insensitive. It is concluded that inhibition of I Cl(Ca) and stimulation of I K(ATP) both contribute to the mechano-inhibitory actions of niflumic acid in the rat portal vein.

Electrical and chemical control of smooth muscle activity of rabbit corpus cavernosum in vitro

This study sought to further elucidate the regulation of cavernous smooth muscle tone and to characterize mechanisms of cavernous activation and relaxation. In isolated strips of rabbit corpus cavernosum, extracellular electrical and mechanical activity were recorded simultaneously before and after pharmacologic stimulation. Spontaneous mechanical activity was characterized by fast phasic contractions (frequency 6 to 30 min-1) associated with fluctuations of the extracellular electrical signals. Phasic activity was increased by blockade of potassium channels or by moderate activation of L-type calcium channels. Faster spikelike fluctuations occurred in the electrical activity, indicating the existence of spike discharges. All mechanical and electrical fluctuations were completely abolished by blockade of L-type calcium channels with nifedipine. Our results indicate that cavernous smooth muscle tone is regulated by both phasic and tonic activation mechanisms caused by the opening of L-type calcium channels and calcium influx through chemically controlled calcium influx/release.

Characteristics of the protective action of alpha-Tocopherol in vascular hypoxia.

The functioning of the guinea-pig isolated portal vein was monitored by measuring spontaneous mechanical activity, responses to electrical stimulation and administered noradrenaline in normoxic conditions. The effect of hypoxia, induced by bubbling the physiological bathing solution with a 95% N(2)/5% CO(2) gas mixture, on the mechanical performance of the vein was then assessed. Spontaneous activity declined in hypoxia, with mean contraction tension reduced by 55 + or - 8.8%. The responses to electrical field stimulation (2-32 Hz, 0.7 msec. 70 V) were lowered by 14 + or - 4.6% but contractions produced by a range of noradrenaline concentrations (0.01-160 mu M) were unaffected by hypoxia. Substitution of glucose in the bathing solution with sucrose, a substrate unavailable to the cells for energy generation, produced a marked enhancement of the effect of hypoxia. Spontaneous activity was reduced by 76 + or - 8.3%, electrically-induced activity by 80 + or - 14.4% and noradrenaline-induced responses by 85 + or - 6.8%. Although in normoxia the activity and responses of the portal vein were unaffected by the presence of alpha-tocopherol, it significantly protected the functioning of the vein in hypoxic conditions. This effect was concentration-dependent within the range 10-160 mu M and was most marked when glucose was replaced by sucrose in the bathing solution.

Effects of polyamines on voltage-activated calcium channels in guinea-pig intestinal smooth muscle.

Effects of polyamines on the spontaneous mechanical and electrical activity of guinea-pig intestinal smooth muscle were studied. Spermine and spermidine inhibited action potential generation and contractions, while putrescine had no effect. Single smooth muscle cells were isolated from the longitudinal muscle layer of the guinea-pig ileum. Whole-cell voltage-clamp experiments were carried out to investigate the effects of polyamines on current through voltage-activated Ca2+ channels. Spermine and spermidine (0.1-1 mM) reduced the inward current in a concentration-dependent manner. Spermine blocked current activated by the dihydropyridine agonist BAY K 8644 (1 microM), whereas no additional inhibition by spermine was seen after blockage of dihydropyridine-sensitive channels by nifedipine (0.1 microM). Inhibition by spermine or spermidine did not shift the peak of the current voltage relation of the inward current. Steady-state activation and inactivation relationships were not affected and thus the amplitude, but not the voltage dependence, of the window current responsible for Ca2+ inflow during sustained depolarization was affected. Putrescine (1 mM) had no significant effect on the inward current. These results suggest that spermine and spermidine inhibit contraction in spontaneously active intestinal smooth muscle by inhibiting Ca2+ current responsible for generation of action potentials.