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Whole-cell patch-clamp recording methods were used to investigate the ionic mechanisms underlying the hyperpolarizing action of galanin in enteric neurones. Galanin suppressed calcium current (ICa) and activated inwardly rectifying potassium current (IK,ir) in AH-type myenteric neurones of guinea-pig small intestine. Both suppression of ICa and activation of IK,ir were concentration-dependent, with an EC50 of 1.4 nmol L-1 and 55 nmol L-1, respectively. Pretreatment with pertussis toxin eliminated both actions of galanin, suggesting that both galanin-induced inhibition of ICa and galanin-induced activation of IK,ir involved activation of Gi/Go proteins. Both suppression of ICa and activation of IK,ir by galanin were mimicked by the N-terminal fragment of galanin, galanin-(1-16) suggesting that the first 16 amino acids of the peptide were sufficient for both actions. The galanin receptor antagonist galantide suppressed the galanin-induced activation of IK,ir with an EC50 of 16 nmol L-1. However, galantide alone suppressed ICa. The results suggest two mechanisms of action for galanin: one is opening of inwardly rectifying potassium channels and the second is blockade of voltage-activated calcium channels.

Perforated patch-clamp methods for recording ionic currents in the whole-cell configuration were used to test the hypothesis that the ionic mechanisms for the excitatory actions of histamine on enteric neurons include suppression of A-type K(+) current (I(A)). Histamine and the selective histamine H(2) receptor agonist, dimaprit, reduced the amplitude of I(A) without affecting the slope factor for I(A) steady-state inactivation curves. Suppression of I(A) was restricted to after hyperpolarization-type myenteric neurons that were immunoreactive for calbindin. The selective histamine H(2) receptor antagonist cimetidine suppressed the action of histamine and dimaprit. Elevation of intraneuronal cAMP by forskolin, a membrane-permeant analog of cAMP, and treatment with a phosphodiesterase inhibitor suppressed I(A.) The results are consistent with the hypothesis that suppression of I(A) is part of the ionic mechanism responsible for elevation of excitability during both slow synaptic excitation and slow synaptic excitation-like responses evoked by paracrine mediators, such as histamine, in after hyperpolarization-type myenteric neurons.

Whole cell perforated patch-clamp methods were used to investigate ionic mechanisms underlying histamine-evoked excitatory responses in small intestinal AH-type myenteric neurons. When physiological concentrations of Na(+), Ca(2+), and Cl(-) were in the bathing medium, application of histamine significantly increased total conductance as determined by stepping to 50 mV from a holding potential of -30 mV. The current reversed at a membrane potential of -30 +/- 5 (SE) mV and current-voltage relations exhibited outward rectification. The reversal potential for the histamine-activated current was unchanged by removal of Na(+) and Ca(2+) from the bathing medium. Reduction of Cl(-) from 155 mM to 55 mM suppressed the current when the neurons were in solutions with depleted Na(+) and Ca(2+). Current-voltage curves in solutions with reduced Cl(-) were linear and the reversal potential was changed from -30 +/- 5 mV to 7 +/- 4 mV. Niflumic acid, but not anthracene-9-carboxylic acid (9-AC) nor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), suppressed the histamine-activated current. A membrane permeable analogue of cAMP evoked currents similar to those activated by histamine. A selective histamine H(2) receptor agonist (dimaprit) mimicked the action of histamine and a selective histamine H(2) receptor antagonist (cimetidine) blocked the conductance increase evoked by histamine. A selective adenosine A(1) receptor agonist (CCPA) reduced the histamine-activated current and a selective adenosine A(1) receptor antagonist (CPT) reversed the inhibitory action. The results suggest that histamine acts at histamine H(2) receptors to increase Cl(-) conductance in AH-type enteric neurons. Cyclic AMP appears to be a second messenger in the signal transduction process. Results with a selective adenosine A(1) receptor agonist and antagonist add to existing evidence for co-coupling of inhibitory adenosine A(1) receptors and histamine H(2) receptors to adenylate cyclase in AH-type enteric neurons.

Biophysical properties of A-type K(+) currents (I(A)) in myenteric neurons from guinea-pig small intestine were studied. I(A) was present in both AH- and S-type myenteric neurons. Reduction of external Ca(2+) did not affect the current. Current density was 13.5+/-10.2 pA/pF in 68 AH-type neurons and 23.4+/-8.2 pA/pF in 31 S-type neurons. S-type neurons appeared to be a homogeneous group based on density of I(A). AH-type neurons were subdivided into two groups with current densities of 9.4+/-4.3 and 25.4+/-4.3 pA/pF. All other biophysical properties of the current were not statistically different for AH- and S-type neurons. Steady-state activation and inactivation curves showed half-activation potentials at -7 mV (k=15. 0 mV) and -86 mV (k=11.5 mV). The curves overlapped at potentials near the resting potential of approximately -55 mV. Time constants for activation ranged from 3.6 to 0.52 ms at test potentials between -20 and 50 mV. Inactivation time constants fell between 41.5 and 11 ms at test potentials between -20 and 50 mV. Time constants for recovery from inactivation fit a double-exponential curve with fast and slow recovery times of 11 and 550 ms. 4-Aminopyridine suppressed I(A) when it was activated at -20 mV following a pre-pulse to -110 mV. Addition of Zn(2+) in the external solution resulted in a concentration-dependent shift of the activation and inactivation curves in the depolarized direction. Zn(2+) slowed the activation and inactivation kinetics of I(A) by factors of 3.3- and 1.2-fold over a wide range of potentials. Elevation of external H(+) suppressed the effect of Zn(2+) with a pK of 7.3-7.4. The effects of Zn(2+) were interpreted as not being due to surface charge screening, because the affinity of Zn(2+) for its binding site on the A-channel was estimated to be between 170 and 312 microM, while the background concentration of Mg(2+) was 10 mM. The enteric nervous system is perceived as an independent integrative nervous system (brain-in-the-gut) that is responsible for local organizational control of motility and secretory patterns of gut behavior. AH- and S-type neurons are synaptically interconnected to form the microcircuits of the enteric nervous system. The results suggest that I(A) is a significant determinant of neuronal excitability for both the firing of nerve impulses and the various synaptic events in the two types of neurons.

Patch-clamp recording was used to study rectifying K+ currents in myenteric neurons in short-term culture. In conditions that suppressed Ca2+ -activated K+ current, three kinds of voltage-activated K+ currents were identified by their voltage range of activation, inactivation, kinetics and pharmacology. These were A-type current, delayed outwardly rectifying current (I(K),dr) and inwardly rectifying current (I(K),ir). I(K),ir consisted of an instantaneous component followed by a time-dependent current that rapidly increased at potentials negative to -80 mV. Time-constant of activation was voltage-dependent with an e-fold decrease for a 31-mV hyperpolarization amounting to a decrease from 800 to 145 ms between -80 and -100 mV. I(K),ir did not inactivate. I(K),ir was abolished in K+ -free solution. Increases in external K+ increased I(K),ir conductance in direct relation to the square root of external K+ concentration. Activation kinetics were accelerated and the activation range shifted to more positive K+ equilibrium potentials. I(K),ir was suppressed by external Cs+ and Ba2+ in a concentration-dependent manner. Ca2+ and Mg+ were less effective than Ba2+. I(K),ir was unaffected by tetraethylammonium ions. I(K),dr was activated at membrane potentials positive to - 30 mV with an e-fold decrease in time-constant of activation from 145 to 16 ms between -20 and 30 mV. It was half-activated at 5 mV and fully activated at 50 mV. Inactivation was indiscernible during 2.5 s test pulses. I(K),dr was suppressed in a concentration-, but not voltage-dependent manner by either tetraethylammonium or 4-aminopyridine and was insensitive to Cs+. The results suggest that I(K),ir may be important in maintaining the high resting membrane potentials found in afterhyperpolarization-type enteric neurons. They also suggest importance of I(K),ir channels in augmentation of the large hyperpolarizing after-potentials in afterhyperpolarization-type neurons and the hyperpolarization associated with inhibitory postsynaptic potentials. I(K),dr in afterhyperpolarization-type enteric neurons has overall kinetics and voltage behaviour like delayed rectifier currents in other excitable cells where the currents can also be distinguished from A-type and Ca2+ -activated K+ current.

Whole-cell perforated patch clamp recordings were used to analyze selectivity of omega-CgTx-MVIIC toxin for voltage-dependent calcium currents in cultured myenteric neurons from guinea-pig small intestine. Omega-CgTx-MVIIC (300 nM) blocked 37 +/- 9% of the peak current activated from -80 mV in 15 neurons by mostly affecting the plateau phase of the current. The toxin suppressed peak current activated from -30 mV dose-dependently with an IC50 of 70 +/- 8 nM. The blockade was complete at toxin concentrations of 1 microM. Thus, it appears that omega-CgTx-MVIIC blocks high voltage activated (HVA) calcium channels in the myenteric neurons unselectively as well as other types of HVA Ca2+ channels including P and Q channels.