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W T Mason

Publications and source records attributed to W T Mason.

At least 73 records · Page 4Linked to original sources
PubMed1989

Dual effects of G-protein activation on Ca-dependent exocytosis in bovine lactotrophs.

The whole-cell patch-clamp technique was used to measure cell membrane capacitance (Cm) to monitor exocytosis in single-cultured bovine prolactin-secreting cells (lactotrophs) of the anterior pituitary. The cells were dialyzed with solutions containing different concentrations of ionised Ca and non-hydrolyzable GTP analogues (GTP-gamma-S and GMP-PNP) to activate G-proteins. We have identified two distinct effects of G-protein activation on Ca-induced exocytosis: (i) the maximum Cm increase due to intracellular Ca-dependent exocytosis was diminished, suggesting an inhibitory role of G-proteins close to the site of granule fusion, while (ii) the rate of Cm increase (delta Cm/delta t) was facilitated, revealing conversely a stimulatory role of G-proteins in the translocation of secretory granules to the fusion sites.

Animals↗
PubMed1989

Beta-adrenergic and opioid receptors on pituicytes cultured from adult rat neurohypophysis: regulation of cell morphology.

Explants of adult rat neurohypophysis were maintained in culture for 14 days. The majority of cells present in the outgrowth of such cultures were identified as pituicytes on the basis of immunostaining for glial fibrillary acidic protein. Pituicytes were also stained by antisera to the membrane glycoprotein antigen Thy-1 and the extracellular matrix glycoprotein fibronectin. The cultures contained naloxone sensitive binding sites for the opioid receptor ligand [3H] dynorphin A 1-8 and peripheral-type benzodiazepine binding sites. Dynorphin binding was visualised over pituicytes following autoradiography. The morphology of cultured pituicytes was regulated by beta-adrenergic receptors present on the cells which, when activated, stimulated rapid transformation from a flattened irregular morphology to a stellate, process-bearing morphology. Dynorphin was without effect on the morphology of cultured pituicytes. These findings are discussed in the context of the known morphological plasticity of pituicytes in vivo.

Animals↗
PubMed1989

Characterization of three types of potassium current in cultured neurones of rat supraoptic nucleus area.

1. Whole-cell, voltage-clamp recordings were obtained from neurones of the supraoptic area of neonatal rats in dissociated cell culture. Recordings were made from neurones having the same morphology as those which were vasopressin or oxytocin immunoreactive. 2. Three types of voltage-activated K+ current were identified on the basis of their kinetics, voltage sensitivities, Ca2+ dependence and pharmacology. The currents corresponded to the delayed rectifier current (IK), the A-current (IA), and the Ca2+-dependent current (IK(Ca] described in other neurones. 3. IK had a threshold of -40 mV, a sigmoidal time course of activation, and was sustained during voltage steps lasting less than 300 ms. The underlying conductance was voltage dependent reaching a maximum at +30 mV (mean maximum conductance 4.09 nS). The activation time constant was also voltage dependent declining exponentially from 4.5 ms at -30 mV to 1.8 ms at +50 mV. 4. IA was transient, and was activated from holding potentials negative to -70 mV; the maximum conductance (mean 5.9 nS) underlying the current was obtained at +10 mV. The activation and inactivation time constants were voltage dependent: the activation time constant declined exponentially between -40 mV (2.2 ms) and +40 mV (0.65 ms). 5. IK and IA were attenuated by the K+ channel blockers tetraethylammonium (TEA) and 4-aminopyridine (4-AP). TEA blocked the conductance underlying IK but appeared to alter the kinetics of IA. In contrast, 4-AP blocked the conductance underlying IA and, to a lesser extent, IK. 6. IK and IA were activated independently of external Ca2+ and the voltage activation of Ca2+ channels since these currents were recorded in the presence of Co2+, a Ca2+ channel blocker. 7. IK(Ca) was recorded only when Ca2+ (2 mM) was present in the external medium. From a holding potential of -30 mV, IK(Ca) had a threshold of -20 mV, was maximal at about +20 mV and declined at more positive potentials. This current was sustained during voltage steps lasting 100 ms and was abolished by addition of Co2+ (2 mM) to the medium. 8. The possible roles of the three K+ currents in regulating the characteristic firing behaviour of supraoptic neurones previously recorded in vivo and in vitro are discussed.

4-Aminopyridine↗
PubMed1989

Characteristics of voltage-gated Ca2+ currents in ovine gonadotrophs.

1. Voltage-clamp recordings were obtained from gonadotrophs of the ovine pars tuberalis in dissociated cell culture, utilizing the whole-cell recording mode of the patch-clamp technique. 2. The amplitudes of Ca2+ and Ba2+ currents were dependent on the extracellular concentration of divalent cation. 3. Ba2+ tail currents were observed on termination of depolarizing voltage steps. The extrapolated amplitudes of 'instantaneous' tail currents increased with membrane depolarization and showed saturation beyond +15 mV. 4. True inactivation of currents occurred in the presence of both external Ca2+ and Ba2+, judged from decrease in tail current amplitudes with progressive increases in duration of the activating voltage pulse. The inactivation process was fitted by a single-exponential function at membrane potentials below -25 mV, while at more depolarized potentials the inactivation was better described by a double-exponential function. The inactivation time constants decreased with positive shifts in membrane potential favouring a voltage-dependent inactivation. 5. The half-value of steady-state inactivation was observed at -40 mV using a two-pulse protocol. 6. Power spectral analysis of Ba2+ current noise from the steady-state portion of inward current showed a double Lorentzian fit of the power spectrum. 7. Two types of voltage-activated Ca2+ currents were identified based on their kinetics, voltage dependence, dependence on activation frequency, differential sensitivity to intracellular ATP and cyclic AMP, and to extracellular application of nifedipine. The channels with faster kinetics had a lower activation threshold (-50 mV) and the amplitude of the current was sensitive to clamping frequency. 8. From ensemble noise analysis of mean maximal inward current, single-channel amplitude of about 1 pA was estimated in 50 mM-Ba2+.

Action Potentials↗
PubMed1988

Choline acetyltransferase immunocytochemical staining of the rat supraoptic nucleus and its surroundings. A light- and electron-microscopic study.

Two different monoclonal antibodies raised against choline acetyltransferase were used, together with preembedding immunocytochemical techniques, to visualize the possible cholinergic innervation of the supraoptic and paraventricular nuclei of the rat hypothalamus. Light microscopy confirmed the presence of a group of bipolar and multipolar immunoreactive neurones in the hypothalamus dorsolateral to the supraoptic nucleus as well as numerous immunopositive fibers. Electron microscopy showed that the immunopositive cell bodies contained the usual perikaryal organelles while most immunoreactive fibers appeared dendritic; immunonegative terminals made synaptic contact onto these profiles. Immunopositive terminals making synaptic contact onto dendritic profiles were also noted in this area. In contrast, light microscopy showed no immunoreactivity to choline acetyltransferase in the magnocellular nuclei themselves. Electron microscopy revealed some immunopositive profiles along the boundaries of both nuclei, along the optic chiasm adjacent to the supraoptic nucleus and in the ventral glial lamina but not within the nuclei proper. Surprisingly, these immunopositive profiles appeared dendritic and were often contacted by one or more immunonegative synapses. Our observations thus indicate that cell bodies and dendrites in the supraoptic and paraventricular nuclei are not directly innervated by cholinergic synapses. The functional significance of the putative cholinergic dendrites in close proximity to magnocellular neurones remains to be determined.

Animals↗
PubMed1988

Ionic currents in cultured supraoptic neurons: actions of peptides and transmitters.

The hypothalamo-neurohypophysial system has proved an excellent model for peptidergic neurons in the central nervous system. Electrophysiological studies using in vivo and in vitro preparations with extracellular and intracellular recording techniques have determined some of the intrinsic and extrinsic mechanisms that generate the striking firing patterns that the neurons exhibit. We have developed a dissociated cell preparation of these neurons and used patch clamp recording techniques to enable detailed studies of membrane properties underlying such activities. Cultured neonatal supraoptic neurons fired spontaneous action potentials which in some cells were distinctively patterned. Under voltage clamp, voltage-activated Na+, K+, and Ca2+ currents were recorded. K+ and Ca2+ currents were modulated by application of alpha-adrenergic agonists, and Ca2+ currents were also modulated by kappa-opioid agonists. The neurons were also sensitive to gamma-aminobutyric acid which acted directly on Cl- channels. Spontaneous, patterned activity, the presence of functional receptors for neurotransmitters and the ability to study the neurons under voltage clamp suggest that this is an excellent model system for studying these peptidergic neurons.

Action Potentials↗
PubMed1988

Characterization of voltage-gated sodium channels in ovine gonadotrophs: relationship to hormone secretion.

1. The properties of whole-cell Na+ currents (INa) were studied in immunocytochemically identified ovine gonadotrophs using the patch clamp technique. 2. Voltage recording under current clamp revealed that gonadotrophs did not fire spontaneously, and fired only a single action potential in response to a depolarizing current clamp step. 3. Under voltage clamp, INa was found to be sensitive to tetrodotoxin (TTX) and had an activation threshold of about -75 mV, with peak current occurring at -20 to -30 mV. 4. Using a two-pulse protocol a delay in the onset of inactivation was observed, suggesting that inactivation is dependent on and preceded by the activation phenomenon. 5. Kinetics of recovery from inactivation of the Na+ channels were studied with test pulses applied at various times after a depolarizing pre-pulse. Recovery from inactivation showed an initial delay, in contrast to the predictions of the Hodgkin-Huxley equations. 6. Recovery from inactivation was examined by using a repetitive pulse protocol, showing approximately 1 s is required for the channels to achieve a 95% recovery. 7. The steady-state inactivation (h infinity -V) curve was sigmoidal and fitted by a logistic growth curve model. The half-inactivation value of the Na+ current occurred at a membrane potential of -70 +/- 8 mV. 8. Noise power spectra derived from fluctuations of INa could be fitted with a single Lorentzian function, and the time constant value was slower at more depolarizing potentials. 9. The single-Na+-channel conductance was estimated from fluctuation analysis under conditions of reduced Na+ current amplitude by depolarizing pre-pulses. The single-channel conductance derived by the above method (approximately equal to 11 pS) corresponded to the single-channel conductance derived from single-channel current measurements using the outside-out version of the patch clamp technique (approximately equal to 13 pS). 10. Inactivation of INa was slowed by including 15 mM-iodate in the pipette. Ensemble fluctuation analysis of INa under these conditions was carried out using the steady state portion of the inactivation phase of the modified INa records, revealing a process best fitted by a double Lorentzian power spectrum, consistent with inactivation kinetics involving both a fast and a slow process. The time constant values correlated well with those obtained from a double-exponential fit to the decaying inactivation phase of the iodate-modified INa.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Whole-cell recordings of ionic currents in bovine somatotrophs and their involvement in growth hormone secretion.

1. The whole-cell mode of the patch-clamp technique was used to record voltage-activated cationic currents in immunocytochemically identified bovine somatotrophs. 2. In current-clamp mode, cells had a resting membrane potential of -83.0 +/- 4.5 mV, and an input resistance of 8.4 +/- 2.1 G omega. Cells rarely fired action potentials spontaneously, but fired one to three action potentials in response to a suprathreshold current pulse. 3. Under voltage clamp, in Ca2+-free media, the action potential was shown to be composed of a TTX-sensitive inward Na+ current and an outward K+ current. 4. The isolated Na+ current had a threshold of approximately -50 mV, and rapidly activated and then inactivated to a small steady-state current. Peak Na+ current amplitude with 140 mM-external Na+ was 341.1 +/- 33.5 pA (n = 14) at a membrane potential of -32.1 +/- 2.4 mV (n = 14). 5. With Ca2+ or Ba2+ (5-30 mM) as the only membrane-permeable cation, voltage pulses to potentials more positive than -55 mV from a holding potential of -80 mV revealed a rapidly activating current component that was followed by a second, very slowly inactivating, current component, most clearly seen with Ba2+. Both components were maximally activated between 0 and +10 mV, were TTX insensitive, but were blocked by 4 mM-Co2+. 6. Three components of the isolated K+ current were identified (IA, IK and IK(Ca] by their voltage sensitivity, Ca2+ dependence and their response to 4-aminopyridine (4-AP) and tetraethylammonium (TEA). 7. Growth hormone (GH)-releasing hormone (GHRH) applied to cells under whole-cell voltage clamp had no effect on either steady-state or voltage-activated ionic currents. This is probably due to dialysis of cytoplasmic compounds vital for GHRH activation of the cell. 8. Both basal and GHRH-stimulated GH secretion were unaffected by TTX, implying that the Na+ action potential is not critical for such release. In contrast the Ca2+ channel blocker Co2+ attenuated GH release in both cases. The K+ channel blocker TEA stimulated GH release above basal and GHRH-stimulated levels.

Action Potentials↗
PubMed1988

Control of secretion in anterior pituitary cells--linking ion channels, messengers and exocytosis.

Normal anterior pituitary cells, in their diversity and heterogeneity, provide a rich source of models for secretory function. However, until recently they have largely been neglected in favour of neoplastic, clonal tumour cell lines of pituitary origin, which have enabled a number of studies on supposedly homogeneous cell types. Because many of these lines appear to lack key peptide and neurotransmitter receptors, as well as being degranulated with accompanying abnormal levels of secretion, we have developed a range of normal primary anterior pituitary cell cultures using dispersion and enrichment techniques. By studying lactotrophs, somatotrophs and gonadotrophs we have revealed a number of possible transduction mechanisms by which receptors for hypothalamic peptides and neurotransmitters may control secretion. In particular, the transduction events controlling secretion from pituitary cells may differ fundamentally from those found in other cell types. Patch-clamp recordings in these various pituitary cell preparations have revealed substantial populations of voltage-dependent Na+, Ca2+ and K+ channels which may support action potentials in these cells. Although activation of these channels may gate Ca2+ entry to the cells under some conditions, our evidence taken with that of other laboratories suggests that peptide-receptor interactions leading to hormone secretion occur independently of significant membrane depolarization. Rather, secretion of hormone and rises in intracellular calcium measured with new probes for intracellular calcium activity, can occur in response to hypothalamic peptide activation in the absence of substantial changes in membrane potential. These changes in intracellular calcium activity almost certainly depend on both intracellular and extracellular calcium sources. In addition, strong evidence of a role for multiple intracellular receptors and modulators in the secretory event suggests we should consider the plasma membrane channels important for regulation of hormone secretion to be predominantly agonist-activated, rather than of the more conventional voltage-dependent type. Likewise, evidence from new methods for recording single ion channels suggests the existence of intracellular sites for channel modulation, implying they too may play an important role in secretory regulation. We shall consider new data and new technology which we hope will provide key answers to the many intriguing questions surrounding the control of pituitary hormone secretion. We shall highlight our work with recordings of single ion channels activated by peptides, and recent experiments using imaging of intracellular ionized free calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Whole cell voltage clamp recordings from cultured neurons of the supraoptic area of neonatal rat hypothalamus.

Whole-cell voltage and current clamp recordings were made from cultured neurons from the hypothalamic supraoptic area of neonatal rats. These neurons fired spontaneous Na+-action potentials, appearing as mixed Na+/K+-currents in voltage clamp. Isolated Na+-currents (less than 3 nA) were rapidly activated and inactivated during positive potential pulses from -80 mV. Two voltage-activated Ba2+-currents (less than 1 nA) were also recorded. These techniques offer a promising new approach for studying the striking electrical behavior of cultured hypothalamic neurons.

Action Potentials↗
PubMed1987

Chloride channels activated by gamma-aminobutyric acid in normal bovine lactotrophs.

The effect of gamma-aminobutyric acid (GABA) on normal bovine lactotrophs was investigated using the patch clamp technique. GABA application (1-10 microM) induced fluctuations of membrane current, accompanied by an increase in inward current. The mean conductance of GABA-activated channels was estimated to be 19 pS from statistical analysis of these fluctuations whereas the mean conductance for GABA-activated single ion channels was 16 pS. The reversal potential of the current was near to the equilibrium potential for chloride ions. These results indicate that the one possible mechanism for inhibition of prolactin release induced by GABA is probably mediated by activation of chloride channels.

Animals↗
PubMed1987

gamma-Aminobutyric acid as an inhibitory neurotransmitter in the rat supraoptic nucleus: intracellular recordings in the hypothalamic slice.

Intracellular recordings have been made from rat supraoptic neurones in the hypothalamic slice preparation. Application of gamma-aminobutyric acid (GABA) caused all neurones to hyperpolarise and this was accompanied by an increase in membrane conductance. GABA application examined on a variety of cells was found to have a potent influence on patterning of electrical activity, always consistent with an inhibitory action.

Acetylcholine↗
PubMed1987

Gamma-aminobutyric acid modulates chloride channel activity in cultured primary bovine lactotrophs.

The effects of GABA, the analogues muscimol and baclofen, and the antagonist bicuculline were investigated on cultured primary bovine lactotrophs using the patch clamp technique. Under voltage clamp in the whole cell mode using solutions containing chloride as the only permeable ion, GABA application increased the amplitude of mean membrane current and fluctuations of current about this mean. Statistical analysis of current fluctuations induced by GABA showed that the power density spectra in 8 of 12 cells were best fitted to double Lorentzian functions and the variance was smallest around the estimated equilibrium potential of chloride ions. The underlying channel open time estimated from noise analysis was only weakly voltage-dependent. The variance of current noise increased with GABA concentration within the range of 1-30 microM, although a slight decrease of variance in one cell could be observed at 30 microM, suggesting that desensitization to GABA might occur. Muscimol mimicked the effect of GABA but baclofen was without effect under these conditions. Bicuculline reduced the GABA-activated membrane current fluctuations. GABA- or muscimol-activated channels recorded in isolated outside-out patches had a slope conductance of about 20 pS. Mean open times of the channel were characterized by two exponential decay functions. We conclude that bovine lactotrophs have GABA-activated chloride channels, which appear to be voltage-independent. In addition, the action of GABA appears to be mediated through the GABAA receptor subtype.

Animals↗
PubMed1987

Voltage-activated currents through calcium channels in normal bovine lactotrophs.

The properties of whole cell Ba2+ currents were studied in immunocytochemically identified, normal bovine lactotrophs using the patch clamp technique. In the current clamp mode, current-induced and spontaneous Ba2+ action potentials were recorded. These were of longer duration and showed less inactivation with stimulation frequency when compared with Na+ action potentials. Under voltage clamp, isolated Ba2+ currents had an activation threshold of about -35 mV and peak value at -15 mV to +20 mV. Inactivation of the current to a potential-dependent, non-zero steady-state level indicated the presence of one rapidly and one slowly inactivating component to the current. These two components were also distinguished by: (1) the voltage dependence of the inactivation time constant of the current, (2) the differential frequency-dependent inactivation of the peak and steady-state currents, and (3) the presence of two half-inactivation potentials for the current. Analysis of the ensemble current variance of the non-inactivating component gave a single-channel amplitude of 0.19 pA at 0 mV and a slope conductance of 3 pS. Fluctuation analysis of the voltage-activated Ba2+ current noise revealed two time constants, one which was voltage dependent and the other was independent of potential. The contribution of these two currents to Ca2+-dependent hormone secretion remains to be clarified.

Action Potentials↗
PubMed1987

Sodium and potassium currents involved in action potential propagation in normal bovine lactotrophs.

1. The properties of whole-cell and single-channel Na+ and K+ currents in immunocytochemically identified bovine lactotrophs were studied using the patch-clamp technique. 2. In the whole-cell, current-clamp mode, cells had membrane potentials of -94.7 +/- 6.7 mV and input resistances of 2-17 G omega. Current-induced action potentials were recorded with a threshold around -35 mV and amplitude of 40-65 mV. Repetitive firing was not sustained at frequencies greater than 1-2 Hz without total inactivation. 3. Under voltage clamp, action potentials were shown to be composed of an inward TTX-sensitive Na+ current and an outward K+ current that was abolished by internal Cs+. 4. The isolated Na+ current had a threshold for activation around -35 mV and rapidly inactivated to a steady state during a test voltage pulse. Inactivation was strongly voltage-dependent, with the Na+ current being half-inactivated at -20 mV. 5. Recovery from inactivation was voltage dependent and at a holding potential of -60 mV, 50% reactivation was achieved after 420 ms. The implications of this long reactivation time on sustained action potential frequency are discussed. 6. Single Na+ channel activity was examined with the outside-out patch configuration and yielded single-channel conductances of 22.5 pS. Reconstruction of the voltage and time dependence of single-channel currents provided an accurate picture of the whole-cell Na+ current. 7. Whole-cell outward current carried by K+ in the absence of Na+ and Ca2+ had a large conductance, was slowly activated and demonstrated no inactivation. A second, more rapidly activating Ca2+-dependent K+ current could also be demonstrated. 8. Ensemble analysis of whole-cell K+ currents in the absence of Ca2+ showed underlying single-channel amplitudes of 1.2 pA at +10 mV, with the lactotroph having about 350 active channels at this potential. 9. Recordings of single K+ channels also demonstrated two classes of channel: a small (50 pS) voltage-activated channel and a higher-conductance (100 pS) Ca2+- sensitive channel. 10. Prolactin secretion was shown to be TTX-insensitive but sensitive to membrane potential, demonstrated as increased release following increased external K+ but not Na+ concentration.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1986

Voltage activated ionic currents in gonadotrophs of the ovine pars tuberalis.

Using whole cell recordings of membrane current and voltage from pars tuberalis gonadotrophs, we have examined the basis of membrane excitability in these cells. This technique allows internal perfusion of gonadotrophs, which has permitted the cell to be filled with CsCl solutions, thus removing outward components of K+ current. Under these conditions, tetrodotoxin (TTX)-sensitive Na+ or TTX-insensitive Ca2+ currents on the order of 500 and 100 pA, respectively, could be detected when the membrane potential was depolarised. Na+ currents, however, were at least 50% inactivated at the normal resting membrane potential of the gonadotroph, and required at least 2 s to recover from inactivation. These data provide the first direct observations of voltage-activated ionic currents in pituitary gonadotrophs, and may explain the TTX insensitivity of LH secretion.

Action Potentials↗
PubMed1986

Single ion channel activity in peptidergic nerve terminals of the isolated rat neurohypophysis related to stimulation of neural stalk axons.

Scanning electron micrographs revealed that the cut face of the sagittally bisected rat neural lobe was characterized by fine fibres bearing multiple rounded swellings (0.5-5 micron in diameter) presumed to be axonal swellings and peptidergic nerve terminals. Patch-clamp electrodes sealed (seal resistance greater than 10(10) omega) on to the cell membrane in such regions revealed (36 out of 43 patches) opening of channels conducting inward current following stimulation of the neural stalk at stimulus frequencies between 5 and 20 Hz. Channel opening did not occur immediately on stimulation (delay 5-15 s) and persisted 1-25 s after stimulation of the axons ceased, implying the possible existence of a depolarization-evoked intracellular messenger. Addition of TTX (5-20 microM) to the bath to block spike propagation abolished stalk stimulation-evoked channel opening but TTX in the patch electrode did not prevent opening. Opening was, however, prevented by addition of 4 mM Co2+ or 4 mM Mn2+. The slope conductance of this channel in medium with 10 mM Ca2+ was approximately 7 pS which was increased to 19 pS in medium containing 95 mM Ba2+. A second inward channel with a slope conductance of 56 pS in medium with 10 mM Ca2+ was also characterized and outward channels were noted in four patches. The electrical properties of nerve terminals are difficult to study because of their small size. The demonstration that ion channel activity can be recorded from mammalian peptidergic nerve terminals thus offers wide scope for future studies on the relation between stimulus and secretion.

Animals↗