Growth Characteristics of Female Radiation/Clinical Oncologists in South Asia: Assessment of Gender Neutrality and Leadership Position.

AIM: To evaluate the temporal growth pattern of female radiation/clinical oncologists (FRCOs) and, if applicable, predict the gender neutrality in different countries of South Asia. MATERIALS AND METHODS: South Asia is composed of Afghanistan, Bhutan, Maldives, Bangladesh, India, Nepal, Pakistan and Sri Lanka. The growth pattern of FRCOs in the latter five countries having radiation oncology facilities was evaluated from respective national registration data. Based on the average annual differential growth rate, together with the already existing female and male radiation/clinical oncologists (MRCOs), the cumulative numbers of FRCOs and MRCOs were forecasted for the next 10 years. The data regarding FRCOs in a leadership position were also calculated from different sources. RESULTS: The total number of radiation/clinical oncologists in the region was 4074, of which 91.8% were in India, because of its vast population. The overall number of FRCOs and MRCOs stood at 1370 and 2704, with a 1:2 female:male ratio. The average incremental annual growth of FRCOs in India was the highest (12.7 persons/year) and Nepal was the lowest (0.4 persons/year), with no data from Pakistan. If the current growth rate is sustained, Indian gender neutrality will be achieved by 2027-2030. In other countries, gender neutrality is unlikely to be achieved in the near future. With regards to leadership positions, 56-77 radiation oncology departments in India, one each in Bangladesh and Sri Lanka are headed by FRCOs, whereas Nepal and Pakistan have none. CONCLUSIONS: With the current growth rate of FRCOs and MRCOs, India will achieve gender parity within a decade; however, the rest of the countries will not achieve this in the near future. Analysis of radiation/clinical oncologists' registration data with their respective national bodies revealed an encouraging growth in the number of FRCOs as against their male counterparts in the last 5 years, compared with previous decades, especially in Bangladesh, Sri Lanka and India. Sri Lanka show high gender neutrality and adopted a multi-tasking and holistic approach of clinical oncology practices as also seen in Scandinavian countries. Such practice may be helpful to improve gender equality in radiation/clinical oncology practice for the other countries in the South Asian region.

Gas spectroscopy through multimode self-mixing in a double-metal terahertz quantum cascade laser.

A multimode self-mixing terahertz-frequency gas absorption spectroscopy is demonstrated based on a quantum cascade laser. A double-metal device configuration is used to expand the laser's frequency tuning range, and a precision-micromachined external waveguide module is used to enhance the optical feedback. Methanol spectra are measured using two laser modes at 3.362 and 3.428 THz, simultaneously, with more than eight absorption peaks resolved over a 17 GHz bandwidth, which provide the noise-equivalent absorption sensitivity of 1.20×10-3 cm-1 Hz-1/2 and 2.08×10-3 cm-1 Hz-1/2, respectively. In contrast to all previous self-mixing spectroscopy, our multimode technique expands the sensing bandwidth and duty cycle significantly.

Dengue in western Terai region of Nepal.

BACKGROUND: Dengue Fever (DF) is an emerging mosquito-borne disease. It is a nagging public health problem in the low lands of Terai, expanding to new areas of Nepal in recent years. METHODS: A cross-sectional study was conducted to determine anti-Dengue IgM positive rate in Mahendranagar, Dhangadi and Dang between August 2008 and November 2009. Serum samples were collected from 283 patients visiting hospitals with history of fever, headache and suspected DF. The samples were examined by ELISA. RESULTS: The anti-Dengue IgM positivity was found to be 9.8 %. The positive rate was highest in Mahendranagar (13.3 %) followed by Dhangadi (9.8 %) (P<0.05). The Dengue positive cases were higher in female (10.9 %) than males (9.0 %). The positivity was higher in Ethnic group Brahman/Chherti (13.1%) as compared to Janajati (5.6 %). The highest positive cases (10.7 %) were from age group above 50 years. The highest numbers of Dengue positive cases were observed in occupation group of agriculture (18.2 %) (P>0.05). CONCLUSIONS: Dengue has substantial expansion in Western and Far Western Terai region of Nepal which was limited to the middle Terai region in the past and mostly infects older people.

Prognostic value of rapid test for diagnosis of dengue in Nepalese patients during 2010 epidemic.

BACKGROUND: Dengue is an emerging vector borne disease in Nepal and rapid diagnostic test is important for early diagnosis of the disease. OBJECTIVES: The aim of the study was to evaluate the diagnostic accuracy of commonly used rapid immunochromatographic test kit in Nepal during 2010 dengue epidemic and to assess disease burden of dengue. METHODS: A total of 131 acute and nonacute serum samples were collected during recent epidemic of dengue in 2010 from clinically suspected Nepalese patients of different hospitals. Rapid immunochromatographic test kit was used for early diagnosis and enzyme immunosorbent was chosen as a reference assay. RESULTS: The sensitivity and specificity of rapid test was 70% and 76.54% respectively whereas the prevalence of the disease was 38.17%. The odds ratio for males was 1.8 however; the association with the disease was statistically not significant. CONCLUSION: The diagnostic accuracy of rapid immunochromatographic test for dengue diagnosis was low (k=0.46). So, it should be substituted by highly sensitive test device for prompt diagnosis and health personnel should consider appropriate timing of sample collection for better performance of rapid test.

Contributions of mature granule cells to structural plasticity in temporal lobe epilepsy.

During the development of epilepsy in adult animals, newly generated granule cells integrate abnormally into the hippocampus. These new cells migrate to ectopic locations in the hilus, develop aberrant basal dendrites, contribute to mossy fiber sprouting, and exhibit changes in apical dendrite structure and dendritic spine number. Mature granule cells do not appear to exhibit migration defects, basal dendrites, and mossy fiber sprouting, but whether they exhibit apical dendrite abnormalities or spine changes is not known. To address these questions, we examined the apical dendritic structure of bromodeoxyuridine (Brdu)-birthdated, green fluorescent protein (GFP)-expressing granule cells born 2 months before pilocarpine-induced status epilepticus. In contrast to immature granule cells, exposing mature granule cells to status epilepticus did not significantly disrupt the branching structure of their apical dendrites. Mature granule cells did, however, exhibit significant reductions in spine density and spine number relative to age-matched cells from control animals. These data demonstrate that while mature granule cells are resistant to developing the gross structural abnormalities exhibited by younger granule cells, they show similar plastic rearrangement of their dendritic spines.

Acute dengue infection in the western terai region of Nepal.

INTRODUCTION: Dengue fever is an emerging mosquito borne disease in Nepal claiming substantial morbidity and mortality. The objective of the study was to find out frequency of acute dengue infection in patients from the hospitals of the western Nepal. METHODS: The study was conducted between August 2007 and July 2008 in patients visiting hospitals of the western terai of Nepal with chief complains of fever. The sero-diagnosis of acute dengue infection was determined by enzyme linked immunosorbent assay among 239 patients visiting Lumbini Zonal Hospital, Butwal; Bheri Zonal Hospital, Nepalgunj; Bardiya District Hospital, Bardiya and Mahakali Zonal Hospital, Mahendranagar. RESULTS: The anti-dengue IgM positivity was 29.3%. There was slight male preponderance with a male to female ratio of 1.2:1. Out of the total positive cases, the highest positive cases (75.7%) were from the age group 15 - 50 years followed by < 15 years old (15.7%). Out of four hospitals, the highest positive cases (54.3%) were in Lumbini Zonal Hospital, Butwal. The age and gender were independent predictors to dengue virus infection. The highest numbers of dengue positive cases were in October (52.6%). The association between dengue disease and the month was statistically significant. CONCLUSIONS: The dengue positivity was estimated in acute patients from hospitals of western Nepal by enzyme immunoassay. Therefore, the serological marker can be used to diagnose acute patients of dengue during outbreaks.

Single ionic channels of two Caenorhabditis elegans chemosensory neurons in native membrane.

The genome of Caenorhabditis elegans contains representatives of the channel families found in both vertebrate and invertebrate nervous systems. However, it lacks the ubiquitous Hodgkin-Huxley Na+ channel that is integral to long-distance signaling in other animals. Nematode neurons are presumed to communicate by electrotonic conduction and graded depolarizations. This fundamental difference in operating principle may require different channel populations to regulate transmission and transmitter release. We have sampled ionic channels from the somata of two chemosensory neurons (AWA and AWC) of C. elegans. A Ca2+-activated, outwardly rectifying channel has a conductance of 67 pS and a reversal potential indicating selectivity for K+. An inwardly rectifying channel is active at potentials more negative than -50 mV. The inward channel is notably flickery even in the absence of divalent cations; this prevented determination of its conductance and reversal potential. Both of these channels were inactive over a range of membrane potentials near the likely cell resting potential; this would account for the region of very high membrane resistance observed in whole-cell recordings. A very-large-conductance (> 100 pS), inwardly rectifying channel may account for channel-like fluctuations seen in whole-cell recordings.

Cyclosporin A protects lung function from hyperoxic damage.

Cyclosporin A (CsA), an inhibitor of protein phosphatase 2B (calcineurin), has been shown to play a role in exocytosis and neutrophil mobility. Hyperoxia (>95% oxygen for 72 h) causes lung injury and reduces lung compliance. This model is indicative of deficiencies in surfactant and elicits a vigorous immune response leading to further damage. We examined the effects of CsA on surfactant-secreting lung alveolar type II cells. CsA enhances ATP-stimulated increases in whole cell capacitance in the presence of 2 mM extracellular Ca2+. This measurement corresponds with increases in exocytosis. Because of its effect on the immune system and exocytosis from type II cells, CsA was examined for its protective effects against hyperoxia-induced lung damage in mice. We found that CsA (50 mg. kg-1. day-1) attenuated hyperoxia-induced reductions in lung compliance when administered before or during 72 h of >95% oxygen (P < 0.05). CsA (10 mg. kg-1. day-1) also had a protective effect against hyperoxia-induced changes in neutrophil infiltration, capillary congestion, edema, and hyaline membrane formation. Wet lung weight-to-dry lung weight ratios did not show any significant changes after hyperoxia or hyperoxia plus CsA (P < 0. 05). CsA may be useful to treat patients undergoing prolonged high-oxygen therapy and possibly other lung injuries.

Adenosine modulates hypoxia-induced responses in rat PC12 cells via the A2A receptor.

1. The present study was undertaken to determine the role of adenosine in mediating the cellular responses to hypoxia in rat phaeochromocytoma (PC12) cells, an oxygen-sensitive clonal cell line. 2. Reverse transcriptase polymerase chain reaction studies revealed that PC12 cells express adenosine deaminase (the first catalysing enzyme of adenosine degradation) and the A2A and A2B adenosine receptors, but not the A1 or A3 adenosine receptors. 3. Whole-cell current- and voltage-clamp experiments showed that adenosine attenuated the hypoxia-induced membrane depolarization. The hypoxia-induced suppression of the voltage-sensitive potassium current (IK(V)) was markedly reduced by adenosine. Furthermore, extracellularly applied adenosine increased the peak amplitudes of IK(V) in a concentration-dependent manner. This increase was blocked by pretreatment not only with a non-specific adenosine receptor antagonist, 8-phenyltheophylline (8-PT), but also with a selective A2A receptor antagonist, ZM241385. 4. Ca2+ imaging studies using fura-2 acetoxymethyl ester (fura-2 AM) revealed that the increase in intracellular free Ca2+ during hypoxic exposure was attenuated significantly by adenosine. Voltage-clamp studies showed that adenosine inhibited the voltage-dependent Ca2+ currents (ICa) in a concentration-dependent fashion. This inhibition was also abolished by both 8-PT and ZM241385. 5. The modulation of both IK(V) and ICa by adenosine was prevented by intracellular application of an inhibitor of protein kinase A (PKA), PKA inhibitor fragment (6-22) amide. In addition, the effect of adenosine on either IK(V) or ICa was absent in PKA-deficient PC12 cells. 6. These results indicate that the modulatory effects of adenosine on the hypoxia-induced membrane responses of PC12 cells are likely to be mediated via activation of the A2A receptor, and that the PKA pathway is required for these modulatory actions. We propose that this modulation serves to regulate membrane excitability in PC12 cells and possibly other oxygen-sensitive cells during hypoxia.

Defective endothelium-dependent relaxation of vascular smooth muscle and endothelial cell Ca2+ signaling in mice lacking sarco(endo)plasmic reticulum Ca2+-ATPase isoform 3.

Sarco(endo)plasmic reticulum Ca2+ ATPase isoform 3 (SERCA3) is one of two Ca2+ pumps serving intracellular Ca2+ signaling pools in non-muscle tissues; however, unlike the ubiquitous SERCA2b, it exhibits a restricted cell-type distribution. Gene targeting was used to generate a mouse with a null mutation in the SERCA3 gene. Homozygous mutant mice were viable, fertile, and did not exhibit an overt disease phenotype. Because SERCA3 is expressed in arterial endothelial cells, aortic ring preparations were analyzed to determine whether it is involved in the regulation of vascular tone. Contraction-isometric force relations in response to phenylephrine or KCl, as well as relaxation produced by exposure to a nitric oxide donor, were similar in wild-type and null mutant aortas. Acetylcholine-induced endothelium-dependent relaxation of aortas after precontraction with phenylephrine was significantly reduced in homozygous mutants (61.3 +/- 5.6% in wild type, 35.4 +/- 7.3% in mutants). Ca2+ imaging of cultured aortic endothelial cells demonstrated that the acetylcholine-induced intracellular Ca2+ signal is sharply diminished in SERCA3-deficient cells and also indicated that replenishment of the acetylcholine-responsive Ca2+ stores is severely impaired. These results indicate that SERCA3 plays a critical role in endothelial cell Ca2+ signaling events involved in nitric oxide-mediated relaxation of vascular smooth muscle.

Loss of protein kinase C inhibition in the beta-T594M variant of the amiloride-sensitive Na+ channel.

We previously reported the presence of a novel variant (beta-T594M) of the amiloride-sensitive Na+ channel (ASSC) in which the threonine residue at position 594 in the beta-subunit has been replaced by a methionine residue. Electrophysiological studies of the ASSC on Epstein-Barr virus (EBV)-transformed lymphocytes carrying this variant showed that the 8-(4-chlorophenylthio) adenosine 3':5'-cyclic monophosphate (8cpt-cAMP)-induced responses were enhanced when compared to wild-type EBV-transformed lymphocytes. Furthermore, in wild-type EBV-transformed cells, the 8cpt-cAMP-induced response was totally blocked by the phorbol ester, phorbol 12-myristate 13-acetate (PMA). This inhibitory effect of PMA was blocked by a protein kinase C inhibitor, chelerythrine. We now have identified individuals who are homozygous for this variant, and showed that PMA had no effect on the 8cpt-cAMP-induced responses in the EBV-transformed lymphocytes from such individuals. Cells heterozygous for this variant showed mixed responses to PMA, with the majority of cells partially inhibited by PMA. Our results demonstrate that an alteration in a single amino acid residue in the beta-subunit of the ASSC can lead to a total loss of inhibition to PMA, and establish the beta-subunit as having an important role in conferring a regulatory effect on the ASSC of lymphocytes.

Norepinephrine-mediated calcium signaling is altered in vascular smooth muscle of diabetic rat.

We studied the influence of diabetes on norepinephrine (NE)-induced changes in intracellular free Ca2+ levels (receptor-mediated Ca2+ signaling) in single tail artery vascular smooth muscle (VSM) cells. VSM cells from 12-16 week streptozotocin-induced diabetic (SID) rats showed an increase in sensitivity to NE when compared to control VSM cells in that the concentration of NE needed to elicit half maximal response of the initial Ca2+ transient was reduced more than 4-fold though the maximal response attained was apparently reduced. In addition, the slope factor (steepness) of the dose-response relation was lowered 4-fold. Moreover, VSM cells of diabetic animals had a higher incidence of NE-induced Ca2+ oscillatory responses. The shift of the dose-response curve to the left, coupled with a higher incidence of oscillations, indicate that the noradrenergic receptor-mediated Ca2+ signaling pathways in tail artery VSM of diabetic rat may be altered.

A novel variant of the beta-subunit of the amiloride-sensitive sodium channel in African Americans.

The amiloride-sensitive sodium channel is responsible for the rate-limiting step of sodium reabsorption in the distal renal tubule, and thus may play a key role in the maintenance of sodium balance and blood pressure. In this study, a genetic variant that results in a change of threonine to methionine at amino acid 594 (T594 M) in the carboxy-terminus of the beta-subunit of the amiloride-sensitive sodium channel has been identified. This variant was present in 6.1% of African-American subjects (N = 231) but was not seen in Caucasians (N = 192). Whole cell voltage clamp of B-lymphocytes from individuals with the T594 M variant showed similar basal membrane slope conductance, compared with the wild-type but increased response to cAMP analog.

Persistence of the olfactory receptor current in a wide variety of extracellular environments.

We measured the current activated by cytoplasmic adenosine 3':5'-cyclic monophosphate (cAMP) in olfactory cilia from the frog Rana pipiens. The odorant-induced current in frog olfactory receptor neurons was also measured for comparison. In both cases, recordings were performed near the neuronal resting potential in a variety of extracellular bath solutions. 2. In Ca(2+)-free baths, cAMP activated an inward current in excised olfactory cilia that was carried entirely by cations. As extracellular Ca2+ was increased, the cationic current decreased while a second current, carried by C1-, increased. Total cAMP-activated current decreased with increasing extracellular CA2+. When external Na+ but not Ca2+ was eliminated, only the C1- component of the current persisted. When external Na+ and Ca2+ were both removed, there was no cAMP-activated current. 3. In receptor neurons, the total odorant-induced receptor current varied in a similar way with the extracellular ionic environment. Under conditions favoring the anionic receptor current, the response amplitude decreased and the latency increased. 4. It is known that olfactory receptor currents persist in a wide variety of extracellular environments. This persistence can be sufficiently explained by the balance between cationic and anionic currents demonstrated here.

Guanine nucleotides modulate steady-state inactivation of voltage-gated sodium channels in frog olfactory receptor neurons.

The voltage for half-inactivation (V1/2) of Na+ currents in frog olfactory receptor neurons (ORNs) under whole-cell voltage clamp showed a shift to more negative potentials with time. Inclusion of guanosine triphosphate (GTP) or its nonhydrolyzable analogue, guanosine-5'-O-3-thiotriphosphate (GTP-gamma-S), which activates G proteins, in the recording pipette, not only gave a more positive V1/2, but also reduced and delayed the negative shift observed in the absence of nucleotides. Guanosine-5'-O-2-thiodiphosphate (GDP-beta-S), a nonhydrolyzable analogue that prevents the binding of GTP to G proteins, did not affect the V1/2 significantly by itself but blocked the positive shift induced by GTP. Since the steady-state activation was not affected, our results indicate that a G protein or a G-protein-dependent process may be important in regulating the steady-state inactivation of Na+ channels in ORNs of the frog.

G-protein activation mediates prepulse facilitation of Ca2+ channel currents in bovine chromaffin cells.

The effects of G-protein activation were investigated on tonic, large depolarization-induced Ca2+ channel facilitation in cultured bovine adrenal chromaffin cells. Under whole-cell voltage clamp, activation of G proteins by intracellular dialysis with 200 microM GTP-gamma S did not significantly affect prepulse facilitation or whole-cell Ba2+ current (IBa) density. In contrast, inactivation of G proteins by intracellular GDP-beta S or pertussis toxin (PTX) pretreatment completely abolished or markedly attenuated facilitation of IBa, respectively. GDP-beta S dialysis resulted in nearly a threefold increase in peak IBa density, whereas PTX pretreatment resulted in a 50% increase. Our results indicate that under control recording conditions (200 microM intracellular GTP), G proteins are tonically activated and suppress high-voltage-activated (HVA) Ca2+ channels in a voltage-dependent and voltage-independent manner. Local superfusion of chromaffin cells with normal bath solution produced a rapid and reversible increase (approximately 50%) in IBa amplitudes that also abolished prepulse facilitation. Together, these results demonstrate that tonic facilitation of HVA Ca2+ channels in bovine chromaffin cells involves the voltage-dependent relief of a G-protein-mediated suppression, imposed by chromaffin cell secretory products that feedback and activate G-protein-coupled autoreceptors.

Angiotensin II suppresses Na+ currents in bovine adrenal chromaffin cells.

We investigated the effects of angiotensin II (ANG II) on the voltage-dependent Na+ channel currents (INa) recorded from bovine adrenal medullary chromaffin cells (BCCs) under whole-cell voltage clamp. Angiotensin II reversibly reduced the peak INa in a dose-dependent fashion. Inhibition was observed at a concentration of 1 nM (6.3 +/- 1.4%, mean +/- SEM) and reached a maximum at 1 microM (35 +/- 3.8%), with a half-maximal effect at 11.6 nM. The ANG II-induced inhibition resulted from a reduction in peak conductance (control, 7.2 +/- 0.7 nS; ANG II 4.3 +/- 0.5 nS; p < 0.01). Angiotensin II had no effect on the reversal potential or the decay time of INa. In addition, the V1/2 and k values, two parameters that describe the voltage dependence of INa for both steady-state activation and inactivation, were not affected by ANG II. The response to ANG II (1 microM) had a delay and attained maximum inhibition in 0.9 +/- 0.2 min (n = 10). Recovery from the effect was slow and took 3.5 +/- 0.8 min (n = 10) after the application of ANG II had been terminated. The inhibitory effects of ANG II were effectively blocked by a specific ANG II receptor antagonist. [Sar1, Val5, Ala8]ANG II. The present study demonstrates that ANG II inhibits voltage-dependent INa+ channel currents in BCCs via a specific receptor-coupled mechanism. The prolonged time course of the ANG II response indicates a possible involvement of second messenger(s) mediating this inhibition.

Activation of serotonin1A receptors inhibits midbrain periaqueductal gray neurons of the rat.

The midbrain periaqueductal gray (PAG) is involved in a variety of functions including pain modulation, vocalization, autonomic control, fear and anxiety. This area contains serotonin receptors, particularly 5-HT1A that are known to play a role in the above functions. The goals of this study were to characterize the effects of 8-OH-DPAT, a selective 5-HT1A agonist, on the firing characteristics and membrane properties of PAG neurons. Both in vivo and in vitro preparations were used. The effects of 8-OH-DPAT on baseline activity of 91 neurons were tested in the in vivo preparation. In 50/91 cells, 8-OH-DPAT produced a decrease in the firing rate that ranged between 21 and 98% (mean +/- S.E.M. decrease of 49 +/- 1.9%). This inhibitory effect was dose dependent and could be blocked by spiperone. In 10/91 cells, 8-OH-DPAT produced an increase in the firing rate that ranged between 13 and 290%, with mean increase of 83 +/- 7.4%. The baseline firing rate of the remaining 31 cells was not affected by 8-OH-DPAT. In the PAG slice preparation, the effects of 8-OH-DPAT on synaptic and membrane properties of 17 PAG neurons were tested using whole-cell voltage clamp-recording procedures. In 14 cells, application of 8-OH-DPAT produced hyperpolarization that ranged between 6 and 21 mV, with mean of 8.4 +/- 2.0 mV. This hyperpolarization was associated with a decrease in membrane impedance that ranged between 8 and 45%, with mean decrease of 21.6 +/- 4.5%. The remaining three neurons did not respond to 8-OH-DPAT.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclic AMP-dependent phosphorylation modifies the gating properties of L-type Ca2+ channels in bovine adrenal chromaffin cells.

We investigated the effects of cAMP-dependent phosphorylation on the voltage- and time-dependent gating properties of Ca2+ channel currents recorded from bovine adrenal chromaffin cells under whole-cell voltage clamp. Extracellular perfusion with the membrane-permeant activator of cAMP-dependent protein kinase, 8-bromo(8-Br)-cAMP (1 mM), caused a 49%, 29%, and 21% increase in Ca2+ current (ICa) amplitudes evoked by voltage steps to 0, +10, and +20 mV respectively (mean values from eight cells, p less than or equal to 0.05). Analysis of voltage-dependent steady-state activation (m infinity) curves revealed a 0.70 +/- 0.27 charge increase in the activation-gate valency (zm) following 8-Br-cAMP perfusion. Similar responses were observed when Ba2+ was the charge carrier, where zm was increased by 1.33 +/- 0.34 charges (n = 8). The membrane potential for half activation (V1/2) was also significantly shifted 6 mV more negative for IBa (mean, n = 8). The time course for IBa (and ICa) activation was well described by second-order m2 kinetics. The derived time constant for activation (tau m) was voltage-dependent, and the tau m/V relation shifted negatively after 8-Br-cAMP treatment. Ca2+ channel gating rates were derived from the tau m and m infinity 2 values according to a Hodgkin-Huxley type m2 activation process. The forward rate (alpha m) for channel activation was increased by 8-Br-cAMP at membrane potentials greater than or equal to 0 mV, and the backward rate (beta m) decreased at potentials less than or equal to + 10 mV. Time-dependent inactivation of ICa consisted of a slowly decaying component (tau h approximately 300 ms) and a "non-inactivating" steady-state component. The currents contributed by the two inactivation processes displayed different voltage dependences, the effects of 8-Br-cAMP being exclusively on the slowly inactivating L-type component.

Single cell assay of exocytosis from adrenal chromaffin cells using "perforated patch recording".

We have combined "perforated patch recording" with phase detection to examine depolarization-induced changes in membrane capacitance (delta Cm) in bovine adrenal chromaffin cells. With this technique, voltage dependent Ca2+ currents and resultant delta Cm's often show little rundown over 1-2 hours even when the free Ca2+ concentration of the pipette is in the millimolar range. By limiting washout of cytosolic components and by maintaining more intact cytosolic Ca2+ buffering, this approach should facilitate the study of stimulus-exocytosis coupling evoked by physiological stimuli which involve cell metabolism and/or membrane receptor triggered second messenger cascades.