Ectopic expression of cone-specific G-protein-coupled receptor kinase GRK7 in zebrafish rods leads to lower photosensitivity and altered responses.

To investigate the roles of G-protein receptor kinases (GRKs) in the light responses of vertebrate photoreceptors, we generated transgenic zebrafish lines, the rods of which express either cone GRK (GRK7) or rod GRK (GRK1) in addition to the endogenous GRK1, and we then measured the electrophysiological characteristics of single-cell responses and the behavioural responses of intact animals. Our study establishes the zebrafish expression system as a convenient platform for the investigation of specific components of the phototransduction cascade. The addition of GRK1 led to minor changes in rod responses. However, exogenous GRK7 in GRK7-tg animals led to lowered rod sensitivity, as occurs in cones, but surprisingly to slower response kinetics. Examination of responses to long series of very dim flashes suggested the possibility that the GRK7-tg rods generated two classes of single-photon response, perhaps corresponding to the interaction of activated rhodopsin with GRK1 (giving a standard response) or with GRK7(giving a very small response). Behavioural measurement of optokinetic responses (OKR) in intact GRK7-tg zebrafish larvae showed that the overall rod visual pathway was less sensitive, in accord with the lowered sensitivity of the rods. These results help provide an understanding for the molecular basis of the electrophysiological differences between cones and rods.

An intermediate conductance K+ channel in the cell membrane of mouse intestinal smooth muscle.

Single channel currents were recorded from cell-attached and inside-out patches in smooth muscle cells of the mouse ileum in order to identify TEA-sensitive Ca2+-dependent K+ channels. Cells were bathed in high-K+ (150 mM) solution with [Ca2+] buffered to 80-150 nM with EGTA and patch pipettes were filled with low-K+ (2.5 mM) physiological solution. Two distinct TEA-sensitive unitary outward current levels were identified at a holding potential (Vh) of 0 mV, corresponding to intermediate conductance (IK, approximately 40 pS) and large conductance (BK, >200 pS) K+ channels. The open probability (Po) of IK channels increased with depolarization, the voltage for half-maximal activation averaging +12 mV in 80 nM Cabath2+. Raising the [Ca2+] in the high-K+ solution from 80 nM to 150 nM increased the Po of IK channels at Vh=0 mV from 0.078 to 0.21. Likewise, the open probability of BK channels at 0 mV was increased from 0.003 to 0.026. Unlike BK channels, IK channels inactivated with maintained depolarization with a voltage for half-maximal inactivation of -66 mV. IK channels were blocked by 2-5 mM external TEA and were sensitive to both charybdotoxin (100 nM) and apamin (500 nM). Our results suggest that IK channels contribute significantly to the Ca2+-dependent K+ conductance in visceral smooth muscle.

Action potential afterdepolarization mediated by a Ca2+-activated cation conductance in myenteric AH neurons.

We investigated the nature of afterdepolarizing potentials in AH neurons from the guinea-pig duodenum using whole-cell patch-clamp recordings in intact myenteric ganglia. Afterdepolarizing potentials were minimally activated following action-potential firing under normal conditions, but after application of charybdotoxin (40 nM) or tetraethyl ammonium (TEA; 10-20 mM) to the bathing solution, prominent afterdepolarizing potentials followed action potentials. The whole-cell current underlying afterdepolarizing potentials (I(ADP)) in the presence of TEA (10-20 mM) reversed at -38 mV and was not voltage-dependent. Reduction of NaCl in the bathing (Krebs) solution to 58 mM shifted the reversal potential of the I(ADP) to -58 mV, suggesting that the current underlying the afterdepolarizing potential was carried by a mixture of cations. The relative contributions of Na(+) and K(+) to this current were estimated to be about 1:5. Substitution of external Na(+) with N-methyl D-glucamine blocked the current while replacement of internal Cl(-) with gluconate did not block the I(ADP). The I(ADP) was also inhibited when CsCl-filled patch pipettes were used. The I(ADP) was blocked or substantially decreased in amplitude in the presence of N-type Ca(2+) channel antagonists, omega-conotoxin GVIA and omega-conotoxin MVIIC, respectively, and was eliminated by external Cd(2+), indicating that it was dependent on Ca(2+) entry. The I(ADP) was also inhibited by ryanodine (10-20 microM), indicating that Ca(2+)-induced Ca(2+) release was involved in its activation. Niflumic acid consistently inhibited the I(ADP) with an IC(50) of 63 microM. Using antibodies against the pore-forming subunits of L-, N- and P/Q-type voltage-gated Ca(2+) channels, we have demonstrated that myenteric AH neurons express N- and P/Q, but not L-type voltage-gated Ca(2+) channels. We conclude that the ADP in myenteric AH neurons, in the presence of an L-type Ca(2+)-channel blocker, is generated by the opening of Ca(2+)-activated non-selective cation channels following action potential-mediated Ca(2+) entry mainly through N-type Ca(2+) channels. Ca(2+) release from ryanodine-sensitive stores triggered by Ca(2+) entry contributes significantly to the activation of this current.

Recording ionic events from cultured, DiI-labelled myenteric neurons in the guinea-pig proximal colon.

To date investigations of enteric neurons by patch clamping/calcium imaging have been limited by studying unidentified heterogeneous populations of neurons. In DiI-labelled colonic myenteric neurons, the feasibility of recording ionic events was determined by applying DiI either to the mucosa or the circular muscle, dispersing neurons after 48 h organotypic culture, and patch-clamping/calcium imaging labeled neurons after 3-7 days in culture. Myenteric neurons with diffuse DiI fluorescence were typically smooth and agranular. Neurons labeled after DiI was applied to circular muscle, fired in either a phasic or a tonic manner, and exhibited fast afterhyperpolarizations (100-300 ms duration) at the end of a depolarizing pulse. They expressed a fast inward current and at least three different outward currents. Action potentials elicited in DiI-labeled sensory neurons were followed by a prolonged afterhyperpolarization (AH, 4-6 s). The offset of a suprathreshold depolarizing step elicited a prolonged outward tail current that approximated the timecourse of the prolonged AH. In addition, in response to membrane depolarization in DiI-labeled neurons loaded with fura-2, robust Ca(2+) transients were recorded using the perforated patch technique. These results demonstrate that DiI labeling of cultured myenteric neurons is feasible, and patch clamp/Ca(2+) fluorescence recordings can be made from specific populations of cultured DiI-labeled colonic myenteric neurons.

Potassium channels in gastrointestinal smooth muscle.

1. Electromechanical coupling in smooth muscle serves to coordinate the contractile activity of the syncytium. Electrical activity of smooth muscle of the gut is generated by ionic conductances that regulate and in turn are regulated by the membrane potential of smooth muscle cells. This activity determines the extent of Ca2+ entry into smooth muscle cells, and thus, the timing and intensity of contractions. 2. Potassium channels play an important role in regulating the excitability of the syncytium. The different types of K+ channel are characterized by different sensitivities to membrane potential, to intracellular Ca2+ levels and to modulation by agonists. 3. This review highlights the different types of K+ channels found in gut smooth muscle and describes their possible roles in regulating the electrical activity of the muscle.

Voltage-dependent large conductance channels in visceral smooth muscle.

The ionic conductances that underlie the resting membrane potential of visceral smooth muscle are not fully understood. Using the patch-clamp technique in the whole-cell configuration, single large conductance channels (LCCs) with unitary conductances of up to 400 pS were recorded in isolated smooth muscle cells of the opossum esophagus. These channels were active at physiological potentials (-100 to -40 mV) and opened with increasing frequency as the membrane potential was hyperpolarized. This voltage dependence gave rise to an inwardly rectifying macroscopic current which was half-maximally activated at -65 mV. The current through LCCs was carried by cations because reduction of external [NaCl] shifted the reversal potential of the LCC current towards the predicted Nernst potential for a nonselective cation current. These results suggest that LCCs may contribute to resting membrane potential in the circular muscle of the opossum esophagus.

Functional innervation of the biliary sphincter of the guinea-pig revealed by anti-autonomic drugs.

1. The roles of excitatory and inhibitory intrinsic motor nerves on contractions reflexly evoked by wall distension were investigated in the isolated sphincter of Oddi of the guinea-pig (SO-GP). 2. Distension of the terminal bile duct for 30-60 s time periods increased the frequency of contractions from about 2 to 12 min(-1) (n = 16). 3. Hexamethonium (HEX; 300 microM) largely prevented the distension-evoked increase in contraction frequency (4.5 min(-1), n = 8) as did atropine (ATR; 1 microM) (0.8 min(-1), n = 6), while tetrodotoxin (TTX; 1 microM) blocked the contractions triggered during distension. 4. L-nitroarginine (L-NA; 100 microM) significantly increased the frequency of contractions during and in the absence distension while apamin (APAM; 0.5 microM) significantly increased their frequency and doubled their mean amplitude during distension. 5. These results suggest that distension activates excitatory cholinergic motor nerves to increase the frequency of contractions in the SO-GP. These actions are modulated by the concomitant activation of intrinsic nitrergic and non-nitrergic inhibitory motor nerves.

Organization of electrical activity in the canine pyloric canal.

1. The electrical activity of the canine gastroduodenal junction was investigated using cross-sectional muscle preparations and intracellular recording techniques. 2. Spontaneous electrical slow waves were recorded from antral and pyloric cells but not from duodenal cells adjacent to the pyloric region. Slow waves were generated in the antrum and propagated to the pyloric region via the circular layer. Pyloric slow waves consisted of an upstroke phase, a plateau phase and oscillations superimposed upon the plateau, whereas antral slow waves had smooth plateau potentials. 3. Within the pylorus slow waves decayed in amplitude with distance from the myenteric border of the circular muscle; the majority of pyloric circular cells were normally electrically quiescent. 4. The longitudinal muscle in the pylorus was electrically coupled and paced by the circular muscle. In longitudinal cells slow waves were usually of long duration with multiple spikes superimposed upon the plateau phase. 5. Nifedipine (10(-8) to 10(-5) M) decreased slow waves amplitude and duration. Tetraethylammonium ions (TEA; 10 mM) increased the duration of slow waves, caused spiking activity during the plateau phase and also elicited spiking in the quiescent regions. 6. The results suggest that gastric slow waves pace the myenteric portion of the circular muscle layer and the longitudinal layer of the pylorus, but do not traverse the gastroduodenal junction, nor pace the majority of cells within the circular muscle of the pylorus. Other excitatory mechanisms are necessary to activate these regions and to co-ordinate their motility with gastric motility.

Suppression of two cloned smooth muscle-derived delayed rectifier potassium channels by cholinergic agonists and phorbol esters.

Functional coupling between muscarinic (m3) receptors and two voltage-gated K+ (Kv) channels (Kv1.2 and Kv1.5) cloned originally from canine colonic smooth muscle was studied using the Xenopus oocytes expression system and a mammalian cell line (COS cells). Oocytes were coinjected with cRNAs encoding the human m3 receptor and the Kv channel clones. COS cells were stably transfected with the hm3 cDNA and the cDNA encoding Kv1.5 channels. In oocytes coexpressing hm3 receptors and Kv channels, acetylcholine (ACh, 100 microM) decreased the whole-oocyte Kv channel current (IKv) by 72% over 20 min. ACh was equally effective at suppressing IKv1.2 as IKv1.5. In oocytes expressing only Kv channels phorbol esters (phorboldibutyrate) and phorbol dideconoate (10-30 nM) mimicked the action of ACh on IKv in oocytes coexpressing hm3 receptors. At the single-channel level, both ACh and phorbol dibutyrate applied to the extra-patch membrane reduced the open probability of Kv channels in the cell-attached patches without affecting single-channel conductance. In cotransfected COS cells, over a similar time course as in oocytes ACh suppressed whole-cell IKv1.5, but only by 30% and the effect was not reversible. These data indicate that stimulation of m3 receptors in cells that express Kv1.2 and Kv1.5 channels causes a poorly reversible decrease in the open probability of these channels.

Identification of single transiently opening ("A-type") K channels in guinea-pig colonic myocytes.

Two K+ channel populations were identified in depolarized cell-attached membrane patches of myocytes freshly dispersed from the circular smooth muscle of guinea-pig proximal colon. First, a large-conductance (150 pS) Ca(2+)-activated K+ channel which was non-inactivating and sensitive to blockade by tetraethylammonium (TEA, 0.5-5 mM); and second, a smaller conductance K+ channel which opened and closed within 100 ms, was insensitive to TEA (0.5-5 mM), but was blocked by 5 mM 4-aminopyridine (4-AP) or maintained depolarization, and which had a unitary conductance of 12-13 pS. The averaged time course of these smaller conductance K+ channels closely resembled the time course of the 4-AP-sensitive, Ca(2+)-insensitive transient outward K+ current recorded in the whole-cell recording mode.

Activation of small conductance Ca(2+)-dependent K+ channels by purinergic agonists in smooth muscle cells of the mouse ileum.

1. Whole-cell and single-channel K+ currents were recorded at room temperature (22-24 degrees C), from smooth muscle cells enzymatically dispersed from the mouse ileum, using variations of the patch-clamp technique. 2. Net outward K+ currents recorded through amphotericin-B-perforated patches in response to step depolarizations positive to -50 mV from a holding potential of -80 mV were decreased by up to 70% by external apamin (0.5 microM). Apamin-sensitive whole-cell currents were also recorded from cells perfused internally with 150 nM Ca2+ but not from cells perfused internally with 85 nM Ca2+. 3. Three types of non-inactivating Ca(2+)-sensitive K+ channels were identified in cell-attached and excised patches under an asymmetrical K+ gradient: (i) large conductance (BKCa; approximately 200 pS) channels blocked by 2 mM external TEA; (ii) intermediate conductance (IKCa; approximately 39 pS) channels blocked by 2 mM external TEA and inhibited by external apamin (0.5 microM); and (iii) small conductance (SKCa; approximately 10 pS) channels that were not blocked by 5 mM external TEA but were sensitive to extracellular apamin (0.5 microM). 4. The TEA-resistant SKCa channels were activated by an increase in [Ca2+]i with an EC50 of 1.5 microM and a Hill coefficient of 1.3. 5. P2 purinoceptor agonists 2-methylthioATP (2-MeSATP), 2-chloroATP and ATP (10-50 microM) increased an apamin-sensitive whole-cell outward K+ current. Extrapatch application of 2-MeSATP (20-100 microM) stimulated the apamin-sensitive IKCa and SKCa channels and activated an apamin-sensitive steady outward current at 0 mV. 6. Smooth muscle cells from the mouse ileum possess two apamin-sensitive K+ channels (IKCa and SKCa); of these, the IKCa channels are TEA sensitive while the SKCa channels are TEA resistant. These channels, along with an apamin-sensitive but TEA-resistant steady outward current, may mediate membrane hyperpolarization elicited by purinergic agonists.

Characterization of ionic currents of circular smooth muscle cells of the canine pyloric sphincter.

1. The ionic currents of circular muscle cells from canine pyloric sphincter were characterized using the whole-cell patch clamp technique. 2. Subpopulations of circular muscle cells from the myenteric and submucosal halves of the circular layer were isolated and studied separately to determine whether differences in the currents expressed by these cells could explain differences in electrical behaviour observed in situ. 3. Resting potentials of isolated cells were about 20 mV positive to cells in intact muscles. Polarization under current clamp to the level of tissue resting potentials caused spontaneous discharge of action potentials in many cells. 4. Outward current measured under voltage clamp could be divided into a voltage-dependent component and a voltage- and Ca(2+)-dependent component. The latter was affected by manipulations of external [Ca2+], nifedipine and dialysis of cells with EGTA. 5. A few cells exhibited a channel that was activated with hyperpolarization. These channels produced inward current at potentials positive to the potassium reversal potential, EK, and reversed at -13 mV. 6. Inward currents, recorded from Cs(+)-loaded cells, were characterized by a transient phase and a sustained phase that persisted throughout the test depolarization. The inward current was reduced by nifedipine but in some cells a nifedipine-resistant component was observed. 7. There were no fundamental differences in the ionic currents recorded from circular muscle cells from the myenteric and submucosal regions, suggesting that the electrical activity of the tissue must be dependent upon structural characteristics (i.e. electrical coupling, fibre bundle dimensions, etc.) of the tissue. 8. The ionic conductance characterized can be related to many of the excitable events recorded from pyloric muscles.

Excitatory and inhibitory neural regulation of canine pyloric smooth muscle.

Studies were performed to characterize the intrinsic innervation of the circular muscle layer of the canine pylorus. Cross-sectional strips of muscle were studied with intracellular recording techniques, and junction potentials were elicited with transmural nerve stimulation. Neurally mediated responses were recorded from cells at several points through the thickness of the circular layer. Excitatory junction potentials (EJPs) increased and inhibitory junction potentials (IJPs) decreased in amplitude with distance from the myenteric border of the circular muscle. Atropine blocked EJPs throughout the circular layer, demonstrating that excitatory inputs are primarily cholinergic. The gradient in IJP amplitude persisted after blockade of EJPs. Three components of IJPs were identified: 1) a fast, apamin-sensitive component that reached a peak and decayed within approximately 1 s; 2) a slower, apamin-insensitive component that reached a peak within 800 ms but decayed slowly over 5 s; and 3) a very slow component that reached a maximum in 7-10 s. Junctional potentials affected the pattern of myogenic electrical activity. Transmural stimulation could evoke premature slow waves in the myenteric portion of the circular layer but when excitatory inputs were blocked, IJPs greatly reduced the amplitude of slow waves. EJPs elicited action potentials in submucosal portion of circular muscles, and IJPs hyperpolarized these cells. The influence of intrinsic nerves on contractile patterns of pyloric muscles was also characterized. These data demonstrate that a neuromuscular apparatus exists within the gastroduodenal junction for 1) local regulation of slow waves and 2) independent control of the myenteric and submucosal regions of the circular layer.

Cholinergic stimulation activates a non-selective cation current in canine pyloric circular muscle cells.

1. Cholinergic stimulation of circular muscle from the canine pyloric sphincter results in excitatory junction potentials and an increase in slow-wave frequency. Experiments were performed on isolated pyloric muscle cells to determine the effects of acetylcholine on membrane conductance and voltage-dependent ionic currents. 2. Acetylcholine depolarized circular muscle cells and increased membrane conductance. Under voltage clamp, these effects were associated with the development of an inward current. 3. The ACh-dependent current (IACh) reversed at about -20 mV and was about equally selective for potassium and sodium. Changes in the chloride gradient had no effect on the reversal potential of IACh. 4. The response to ACh was blocked by atropine suggesting that the response was mediated by muscarinic receptors. IACh could not be elicited in the presence of ions normally used to block potassium currents (e.g. bath-applied TEA+ and replacement of Ki+ with Csi+. 5. In some cells single-channel openings could be resolved in response to ACh. These channels had a slope conductance of 30 pS, and open probability increased with depolarization. 6. Acetylcholine had little or no effect on voltage-dependent Ca2+ currents, and increased voltage-dependent outward currents. The latter effect may have been due to increased release of Ca2+ from internal stores. 7. The non-selective cationic current elicited by ACh can explain the excitatory junction potentials in pyloric muscle cells that are generated by transmural nerve stimulation and may also explain the chronotropic effects of ACh on slow waves.

Cholinergic nerve-mediated excitation in the guinea pig choledochoduodenal junction activated by distension.

The electrical basis of propulsive contractions in the guinea pig choledochoduodenal junction (CDJ), which are triggered by distension, was investigated using intracellular microelectrode recording techniques. The isolated CDJ was placed in a continuously perfused tissue chamber at 37 degrees C. Membrane potential was recorded from smooth muscle cells in either the ampulla or in the upper CDJ (upper junction) regions, which were immobilized by pinning. Distension of the upper junction (20-30 s) by increasing intraductal hydrostatic pressure (mean elevation: 2.0 +/- 0.3 kPa, n = 13) triggered "transient depolarizations" (TDs: < 5 mV in amplitude and 2-5 s in duration) and action potentials in the circular muscle layer of the ampulla. The frequency of TDs in the ampulla was increased from 2.2 +/- 0.2 to 15.9 +/- 2.2 min-1 (n = 13) during distension. Simultaneous impalements of cells in the longitudinal and circular muscle layers in the ampulla revealed that subthreshold TDs in the circular layer were associated with an increased rate of action potential discharge in the longitudinal layer. Atropine (Atr; 1.4 x 10(-6) M) and tetrodotoxin (TTX; 3.1 x 10(-6) M blocked the distension-evoked increase in TD frequency, without affecting the frequency of ongoing TDs. The sulfated octapeptide of cholecystokinin (1-5 x 10(-8) M) increased the amplitude of TDs recorded in the circular muscle layer of the ampulla and increased action potential discharge rate. In separate recordings, radial stretch of the ampulla region increased the rate of discharge of action potentials in the smooth muscle of the upper junction.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation between electrical and morphological properties of canine pyloric circular muscle.

Electrical slow waves decay in amplitude as they conduct from the myenteric to the submucosal regions of the circular muscle layer in the canine pyloric sphincter. We used the partitioned chamber method to study the passive and active properties of pyloric muscles, and we found that length constants of circular muscles of myenteric region were significantly longer than muscles near the submucosal surface. These data suggested differences in either membrane resistance, junctional resistance, or cytoplasmic resistance. The first parameter was evaluated by measuring time constants in intact tissues and single cells isolated from the submucosal and myenteric regions. Membrane time constants were not different in the two regions, nor were differences found in the input resistances of isolated cells. Morphological studies failed to demonstrate differences in cell diameters in the two regions suggesting that cytoplasmic resistances are similar. These findings suggest that the different cable properties in the two regions may be due to differences in electrical coupling. Morphological examination revealed similar numbers of gap junctions between cells in the two regions, but large differences were noted in the size of muscular bundles. Muscles of the myenteric region were arranged into large, tightly packed bundles, whereas muscles of the submucosal region consisted of small bundles with an extensive extracellular space filled with connective tissue. We suggest that the difference in cable properties may be due to differences in electrical coupling between bundles. These data suggest that submucosal muscles function more like a multiunit smooth muscle, whereas myenteric muscles behave as a single unit.

Suppression of a slow post-spike afterhyperpolarization by calcineurin inhibitors.

A subset of myenteric neurons in the intestine (AH neurons) generate prolonged (>5 s) post-spike afterhyperpolarizations (slow AHPs) that are insensitive to apamin and tetraethylammonium. Generation of slow AHPs depends critically on Ca(2+) entry and intracellular release of Ca(2+) from stores, which then leads to the activation of a K(+) conductance that underlies the slow AHP (g(sAHP)). Slow AHPs are inhibited by stimulation of the cAMP/protein kinase A (PKA) pathway, suggesting that phosphorylation of the K(+)-channels that mediate the g(sAHP) (K(sAHP)-channels) is responsible for suppression of slow AHPs and possibly for the repolarization phase of slow AHPs. In the present study, we investigated the possibility that the rising phase of the slow AHP is mediated by dephosphorylation of K(sAHP)-channels by calcineurin (CaN), a Ca(2+)-calmodulin-dependent protein phosphatase, leading to an increase in g(sAHP) and activation of the associated current I(sAHP). Slow AHPs and I(sAHP) were recorded using conventional recording techniques, and we tested the actions of two inhibitors of CaN, FK506 and cyclosporin A, and also the effect of the CaN autoinhibitory peptide applied intracellularly, on these events. We report here that all three treatments inhibited the slow AHP and I(sAHP) (>70%) without significantly affecting the ability of neurons to fire action potentials. In addition, the slow AHP and I(sAHP) were suppressed by okadaic acid, an inhibitor of protein phosphatases 1 and 2A. Our results indicate that activation of the g(sAHP) that underlies the post-depolarization slow AHPs in AH neurons is mediated by the actions CaN and non-Ca(2+)-dependent phosphatases.

Patterns of electrical activity and neural responses in canine proximal duodenum.

The patterns of electrical activity and neural inputs to the proximal duodenum between the pyloric sphincter and the sphincter of Oddi were studied in muscles of the dog. Smooth muscle cells in the most proximal region were electrically quiescent, but slow waves were recorded in all regions distal to the first few millimeters. Electrical activity was recorded from circular muscle cells near the myenteric and submucosal surfaces of the circular layer, and slow wave activity was similar in both regions. The nature of neural inputs was also characterized. With electrical field stimulation, responses in cells near the submucosal surface were predominantly excitatory junction potentials (EJPs); near the myenteric border responses were either inhibitory junction potentials (IJPs) or biphasic responses (i.e., small EJPs followed by IJPs). EJPs were blocked by atropine. IJPs were nonadrenergic and noncholinergic (NANC), and several experiments suggested that nitric oxide (NO), or a NO-releasing compound, serves as the inhibitory neurotransmitter in this region. Exogenous NO caused hyperpolarization of membrane potential. IJPs and the hyperpolarization response to NO were sensitive to apamin. These data describe the myogenic mechanisms and neurogenic apparatus that appear to regulate motility in the most proximal region of the duodenum.

Regulation of calcium current by voltage and cytoplasmic calcium in canine gastric smooth muscle.

The regulation of Ca2+ current by intracellular Ca2+ was studied in isolated myocytes from the circular layer of canine gastric antrum. Ca2+ current was measured with the whole cell patch-clamp technique, and changes in cytoplasmic Ca2+ ([Ca2+]i) were simultaneously measured with indo-1 fluorescence. Ca2+ currents were activated by depolarization and inactivated despite maintained depolarization. Ca2+ current inactivation was fit with a double exponential function. Using Ba2+ or Na+ as charge carriers removed the fast component of inactivation, whereas enhanced intracellular buffering of Ca2+ did not remove the fast component. Ca2+ currents were associated with a rise in [Ca2+]i. The decrease in [Ca2+]i following repolarization was exponential, and during the relaxation of [Ca2+]i, Ca2+ current was inactivated. The inward current recovered with a similar time course as the decrease in [Ca2+]i, suggesting that [Ca2+]i regulates the basal availability of Ca2+ channels. These data support the hypothesis that, although [Ca2+]i may influence the resting level of inactivation, it is the "submembrane" compartment of [Ca2+]i that regulates the development of inactivation.

Effects of nerve stimulation on the spontaneous action potentials recorded in the proximal renal pelvis of the guinea-pig.

The effects of nerve stimulation on the electrical and mechanical activity of the smooth muscle of the proximal renal pelvis of the guinea-pig were investigated using standard tension and microelectrode recording techniques. Spontaneous action potentials were deemed to have been recorded from three cell types: (1) "pacemaker" cells (9 of > 120) had membrane potentials (MPs) of -42.1 +/- 2.9 mV and fired action potentials of a simple waveform; (2) "driven" cells (> 100) had more stable MPs of -56.1 +/- 1.2 mV (n = 36) and more complex "ureter-like" action potentials; (3) the remaining cells had MPs of -45.5 +/- 1.7 mV (n = 15) and action potentials with a waveform "intermediate" to groups (1) and (2). Nifedipine (0.1-1 microM) and Cd2+ (0.1-1 mM) blocked all spontaneous action potential discharge and depolarized the membrane to near -40 mV. Intramural nerve stimulation (10-50 Hz for 1-10 s) increased both the amplitude and frequency of the spontaneous contractile activity, this increase peaked in about 30 s and decayed slowly over several minutes. Nerve stimulation depolarized pacemaker and driven cells 9.1 +/- 3.5 (n = 3) and 1.6 +/- 0.7 (n = 6) mV, respectively; the frequency of their action potential discharge increased from 7.6 +/- 2.7 and 9.9 +/- 1.1/min to 17.3 +/- 0.5 and 11.1 +/- 1.4/min, respectively. The duration of the action potentials in driven cells also increased significantly for several minutes. All these effects were blocked by tetrodotoxin (TTX) (1.6 microM). It was concluded that the positive chronotropic and inotropic effects of nerve stimulation on renal pelvis contractility can be correlated with the changes in the frequency and duration of the action potentials recorded in driven cells.