SUMMARY1. The effects of somatostatin and somatostatin analogues on a Ca2+ current from acutely isolated and short-term (24-48 h) cultured adult rat superior cervical ganglion (SCG) neurones were studied using the whole-cell variant of the patchclamp technique.2. Somatostatin (SOM) produced a rapid, reversible and concentrationdependent reduction of the Ca2+ current. Ca2+ current amplitude was reduced over the voltage range -15 to + 40 mV with the greatest reduction occurring where the amplitude was maximal (ca + 10 mV). In the presence of SOM, the Ca2+ current rising phase was slower and biphasic at potentials between 0 and +40 mV.3. Application of 0 1 ,tM-SOM for greater than 10 s resulted in a desensitization of the response. During a 4 min application of 01I aM-SOM, Ca2+ current amplitude returned to about 90 % of control. A second application of 0 1 ,IM-SOM produced less block than the initial application.4. Concentration-response curves for SOM, somatostatin-14 (SOM-14) and somatostatin-28 (SOM-28) were fitted to a single-site binding isotherm. The concentrations producing half-maximal block and the maximal attainable blocks of the Ca2+ current for SOM, SOM-14 and SOM-28 were 3 3, 5 4 and 35 nm, respectively and 55, 51 and 54%, respectively. SOM-14 and SOM-28 slowed the Ca2+ current rising phase in a manner similar to that of SOM. Somatostatin-28[1 12] had no effect on the Ca2+ current at 1 JIM. S. R. IKEDA AND G. G. SCHOFIELD significantly less block of Ca2+ current amplitude (7-1 and 14-7 %, respectively) when compared with controls.7. Internal dialysis with solutions containing 500 /M-guanosine-5'-0-(2-thiodiphosphate)(GDP-,8-S) had no significant effect on either the Ca21 current amplitude or block produced by 01 ,sM-SOM.8. Internal dialysis with solutions containing 500 /LM-cyclic adenosine 3',5'-monophosphate (cyclic AMP) and 3-isobutyl-1-methylxanthine had no significant effect on either the Ca2+ current block produced by 0-1 /tM-SOM or the Ca2+ current amplitude. 9. Treatment of SCG neurones in short-term culture with 200 ng/ml pertussis toxin (PTX) for 12-16 h greatly reduced the ability of 01 #,M-SOM to produce Ca2+ current block. Mean Ca2+ current block was 4-9 and 40-8% for PTX-treated and control cultures; respectively. 10. These results suggest that somatostatin blocks a Ca2+ current in adult rat SCG neurones via a pertussis toxin-sensitive guanine nucleotide regulatory protein. This effect appears to be independent of intracellular cyclic AMP.
The electrical properties of nodose ganglion cells acutely isolated from adult rats were studied using the whole-cell patch-clamp recording method. Current-clamp recordings revealed a mean resting membrane potential of -54.3 mV and an input resistance of 527 M omega. Depolarizing current steps evoked action potentials with the following properties (mean): amplitude 111 mV, threshold -36 mV, and rate of rise 117 V/s. Two types of action potentials were observed, short and long duration. These properties, with the exception of input resistance (527 M omega cf. 50 M omega), are similar to those reported previously using intracellular recording methods in intact nodose ganglia (11, 20, 28). Brief application of 10 microM 5-hydroxytryptamine resulted in a rapid depolarization and burst of action potentials in the majority of cells. With voltage-clamp recording, step depolarizations to potentials positive to -10 mV elicited a transient inward current that was followed by a sustained outward current. Inward Na+ current was isolated by ion substitution and pharmacological agents. Two types of Na+ current were observed. One current was completely abolished by 3-15 microM tetrodotoxin (TTX), had a rapid time course, activated over the potential range -60 to -10 mV, and attained half-maximal conductance at -30 mV. The other current persisted in the presence of 15 microM TTX, had a slower time course, activated over the potential range -30 to 0 mV, and attained half-maximal conductance at -15 mV. In addition, 500 microM Cd2+ and 5.0 mM Co2+ reduced the TTX-insensitive current to 53 and 42% of control, respectively. Inward Ca2+ current was isolated by ion substitution and pharmacological agents and was identified by a dependence on external Ca2+. Cd2+ (500 microM) and Co2+ (5 mM) reduced the maximal inward current to 5 and 20% of control, respectively. When Ba2+ was substituted for Ca2+ as the charge carrier, the maximal inward current increased to 175% of control. Some cells had two Ca2+ current components, an inactivating component that activated near -60 mV and a large sustained current that activated near -40 mV. The initial inactivating current appeared as a "hump" on the current-voltage (I-V) curve over the potential range of -60 to -30 mV. The results indicate that, following isolation of these adult mammalian neurons, the membrane surfaces are sufficiently clean to allow patch-clamp recording.(ABSTRACT TRUNCATED AT 400 WORDS)
Neurons enzymatically isolated from the adult rat superior cervical ganglion (SCG) were investigated using the whole-cell variant of the patch-clamp technique. Current-clamp studies revealed the following mean passive and active membrane properties: resting membrane potential, -54.9 mV; input resistance, 349 M omega; action potential (AP) threshold, -29.8 mV; AP overshoot, 53.3 mV; AP maximum rate of rise, 166.4 V/s; and AP duration, 3.2 ms. Chemosensitivity to acetylcholine remained intact following enzymatic dispersion. Voltage-clamp studies of a transient tetrodotoxin-sensitive Na+ current revealed activation and inactivation processes which could be fit to modified Boltzmann equations. Na+ current activation parameters for the half activation potential (Vh) and slope factor (K) were -23.3 mV and 5.3 mV, respectively. Inactivation parameters for Vh and K were -59.3 mV and 7.6 mV, respectively. Voltage-clamp studies also revealed a high voltage-activated sustained inward current which was eliminated upon removal of external Ca2+, greatly reduced by 500 microM Cd2+, and supported by Ba2+ or Sr2+. Tail current analysis of this Ca2+ current revealed a sigmoidal activation. A low voltage-activated transient Ca2+ current was not observed. We conclude that isolated SCG neurons retain the properties of neurons in intact ganglia and provide several advantages over conventional preparations for the study of voltage-gated membrane currents.
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