M. Lamacz

Patch-Clamp Study of the Ionic Currents Underlying Action Potentials in Cultured Frog Pituitary Melanotrophs

The ionic conductance mechanisms underlying the action potential behaviour of frog melanotrophs in primary culture were studied by using the patch-clamp technique in whole-cell configuration. The action potentials spontaneously generated by these cells were predominantly sodium spikes with a calcium component. Voltage-dependent sodium, calcium, potassium and calcium-activated potassium currents were identified and analysed separately. The voltage-dependent sodium current was characterized by its fast kinetic, its low-threshold activation, its voltage-dependent inactivation and a tetrodotoxin sensitivity. Calcium currents were identified on the basis of their ionic selectivity to divalent cations (Ba2+, Ca2+, Co2+) and their time course. Only two of the three well-documented calcium currents could be detected in frog melanotrophs. A sustained calcium current (ICaS) and an inactivating calcium current (ICaN) were elicited by step depolarizations up to -20 mV. ICaN inactivated for membrane potentials more positive than -50 mV; its inactivation appeared to be both voltage- and calcium-dependent. Transient calcium current (ICaT) has never been observed. Two types of potassium currents were identified: voltage-dependent potassium (IKV) and calcium-activated potassium currents, (IK[Ca]). They were both suppressed by tetraethylammonium chloride, whereas only IK(Ca) was blocked by cobalt. These major ionic currents underlying spontaneous electrical activity are assumed to be involved in the process of alpha-melanocyte-stimulating hormone release. The present study provides the ground for future investigations regarding the relationships between the electrical and secretory activities in amphibian pars intermedia cells.

The endozepine ODN stimulates polyphosphoinositide metabolism in rat astrocytes

Astrocytes synthesize a series of peptides called endozepines which act as endogenous ligands of benzodiazepine receptors. The present study demonstrates that the endozepine ODN causes a dose‐dependent increase in inositol trisphosphate and a parallel decrease in phosphatidylinositol bisphosphate in cultured rat astrocytes. Pre‐incubation of astrocytes with the phospholipase C inhibitor U 73122 or with pertussis toxin totally blocked polyphosphoinositide metabolism. These data show that, in rat astrocytes, ODN stimulates a phospholipase C coupled to a pertussis toxin‐sensitive G protein.

V. Melanotropin release inhibiting activity of neuropeptide Y: Structure-activity relationships

Abstract We have recently shown that the release of α-MSH by the intermediate lobe of the frog pituitary is inhibited by neuropeptide Y (NPY). Using the perifusion technique, we have compared in the present study, the α-MSH release inhibiting activities of NPY, various NPY short chain analogues and two other members of the pancreatic polypeptide family, peptide YY (PYY) and avian pancreatic polypeptide (APP). The order of biological potency was NPY > NPY[2–36] > NPY[16–36] > NPY[25–36] > NPY[1–15]. Among the two pancreatic polypeptides tested, PYY appeared to be almost as potent as NPY while APP was 6 times less active than NPY. Neither NPY[1–15] nor NPY[16–36] could antagonize the inhibitory effect of NPY on α-MSH release. The structure-activity relationship study suggests that the bioactive determinant of NPY is located in the C-terminal part of the molecule.

The endogenous benzodiazepine receptor ligand ODN increases cytosolic calcium in cultured rat astrocytes

We have investigated the production of diazepam-binding inhibitor (DBI)-related peptides by astrocytes in primary culture and we have determined the effect of the octadecaneuropeptide DBI[33-50] (ODN) on the intracellular calcium concentration ([Ca2+]i) in astrocytes. Immunocytochemical labeling with antibodies against ODN showed that cultured astrocytes retain their ability to synthesize DBI in vitro. Cultured astrocytes were also found to release substantial amounts of ODN-immunoreactive material, and a brief exposure of astrocytes to a depolarizing potassium concentration resulted in a 5-fold increase in the rate of release of the ODN-like peptide. Microfluorimetric measurement of [Ca2+]i with the fluorescent probe indo-1 showed that nanomolar concentrations of ODN induced a marked increase in [Ca2+]i. The stimulatory effect of ODN on [Ca2+]i was not affected by calcium channel blockers or by incubation in Ca(2+)-free medium. In contrast, thapsigargin, an inhibitor of microsomal Ca(2+)-ATPase activity, totally abolished the ODN-induced increase in [Ca2+]i. Repeated pulses of ODN caused attenuation of the response, indicating the existence of a desensitization phenomenon. Preincubation of astrocytes with pertussis toxin totally blocked the effect of ODN on [Ca2+]i. The present study indicates that ODN-related peptides are synthesized and released by glial cells. Our results also show that synthetic ODN induces calcium mobilization from an intracellular store through stimulation of pertussis toxin-sensitive G protein. Taken together, these data suggest that endozepines act as paracrine and/or autocrine factors controlling the activity of astroglial cells.

Multihormonal Regulation of Pituitary Melanotrophs

The pars intermedia of the pituitary is composed of one predominant category of endocrine cells, the melanotrophs, which synthesize the precursor protein proopiomelanocortin (POMC). 1,2 In lower vertebrates a-melanocyte stimulating hormone (a-MSH), which plays a pivotal role in skin color adaptation, is considered as the major peptide hormone secreted by melanotrope cells. Early experiments demonstrated that the release of MSH from the intermediate lobe is primarily under an inhibitory control from the hypothalamus. In particular, hypoth~amic lesions, pituitary stalk section, and autotransplantation of the pituitary all induce darkening of the animal^.^.^ However, Wilson and Morgan have observed that the concentration of MSH in the plasma of black-adapted Xenopus laevis was higher than in pituitary stalk-sectioned animals,’ suggesting the involvement of stimulatory hypothalamic factor(s) in the control of MSH release. The purpose of the present review is to summarize our current knowledge on the regulatory mechanisms that are involved in the control of the activity of amphibian pituitary meianotrophs.

Involvement of extracellular and intracellular calcium sources in TRH-induced α-MSH secretion from frog melanotrope cells

The stimulatory effect of thyrotropin-releasing hormone (TRH) on alpha-melanocyte stimulating hormone (MSH) secretion from the frog pars intermedia is mediated through the phospholipase C (PLC) pathway but requires extracellular Ca2+. The aim of the present study was to investigate the respective contribution of extracellular and intracellular Ca2+ in the action of TRH on cytosolic calcium concentration ([Ca2+]i) and alpha-MSH release. In normal conditions, TRH (10(-7) M; 5 s) evoked two types of Ca2+ responses: in 63% of the cells, TRH caused a sustained and biphasic increase in [Ca2+]i while in 37% of the cells, TRH only induced a transient response. In the presence of EGTA or Ni2+, the stimulatory effect of TRH on [Ca2+]i and alpha-MSH secretion was totally suppressed. Nifedipine (10(-6) M) reduced by approximately 50% the amplitude of the two types of Ca2+ responses whereas omega-conotoxin GVIA (10(-7) M) suppressed the plateau-phase of the sustained response indicating that the activation of L-type Ca2+-channels (LCC) is required for initiation of the Ca2+ response while N-type Ca2+-channels (NCC) are involved in the second phase of the response. Paradoxically, neither nifedipine nor omega-conotoxin GVIA had any effect on TRH-induced alpha-MSH secretion. The PLC inhibitor U-73122 (10(-6) M) significantly reduced the transient increase in [Ca2+]i and totally suppressed the sustained phase of the Ca2+ response but had no effect on TRH-induced alpha-MSH secretion. The stimulatory effect of TRH on PLC activity was not effected by nifedipine and omega-conotoxin GVIA but was abolished in Ca2+-free medium. Ryanodine had no effect on the TRH-induced stimulation of [Ca2+]i and alpha-MSH secretion. Concomitant administration of nifedipine/omega-conotoxin GVIA or U-73122/omega-conotoxin GVIA markedly reduced the response to TRH but did not affect TRH-evoked alpha-MSH release. In contrast, concomitant administration of U-73122 and nifedipine significantly reduced the effect of TRH on both [Ca2+]i and alpha-MSH release. Taken together, these data indicate that, in melanotrope cells, activation of TRH receptors induces an initial Ca2+ influx through nifedipine- and omega-conotoxin-insensitive, Ni2+-sensitive Ca2+-channels which subsequently activates LCC and causes Ca2+ mobilization from intracellular pools by enhancing PLC activity. Activation of the PLC causes Ca2+ entry through NCC which is responsible for the plateau-phase of sustained Ca2+ response. Although nifedipine and U-73122, separately used, were devoid of effect on secretory response, Ca2+ entry through LCC and mobilization of intracellular Ca2+ are both involved in TRH-evoked alpha-MSH release because only one source of Ca2+ is sufficient for inducing maximal hormone release. In contrast, the Ca2+ influx through NCC does not contribute to TRH-induced alpha-MSH secretion.

In vitro study of frog (Rana ridibunda Pallas) neurointermediate lobe secretion by use of a simplified perifusion system: IV. Interaction between dopamine and thyrotropin-releasing hormone on α-melanocyte-stimulating hormone secretion

The interaction between dopamine and TRH on alpha-melanocyte-stimulating hormone (MSH) release from the intermediate lobe of amphibian pituitary has been studied in vitro using the perifusion model. Dopamine (10(-10) to 10(-6) M) was responsible for a dose-related inhibition of alpha-MSH secretion. The inhibitory effect of dopamine (10(-8) and 3.16 X 10(-8) M) was completely abolished in the presence of haloperidol (10(-5) and 10(-6) M, respectively). It has been previously established that, in amphibians, TRH stimulated alpha-MSH release in vitro and that the action of TRH was not mediated via an inhibition of the release of endogenous dopamine (M. C. Tonon, P. Leroux, M. E. Stoeckel, S. Jégou, G. Pelletier, and H. Vaudry, 1986, Endocrinology 112, 133-141). In the present study we demonstrate that TRH (10(-7) M) reverses the inhibitory effect of dopamine (for concentrations ranging from 3.16 X 10(-8) to 10(-6) M) on alpha-MSH secretion and that the effects of TRH and dopamine are additive. Thus, these results indicate that the intracellular events associated with TRH-induced stimulation and dopamine-induced inhibition of alpha-MSH release are not linked together.

II. Atrial natriuretic factor (ANF) stimulates the release of α-MSH from frog neuro- intermediate lobes in vitro. Interaction with dopamine, GABA and neuropeptide Y.

Abstract The action of atrial natriuretic factor (ANF) on α-MSH release from frog neurointermediate lobe was studied in vitro using a perifusion technique. Synthetic ANF Arg101Tyr126, at concentrations ranging from 10 −7 to 10 −5 M, caused a dose-related stimulation of α-MSH release. In addition, dopamine, GABA and NPY, three neuroendocrine factors which inhibit α-MSH secretion totally suppressed the action of ANF on α-MSH production. The neural lobe of the amphibian pituitary contains numerous ANF immunoreactive fibers, and this regulatory peptide may diffuse from nerves terminating in the pars nervosa to the pars intermedia. Thus, our results suggest that ANF of hypothalamo-neurohypophysial origin may be involved in the multineuronal regulation of amphibian melanotrophs.

Effect of acetylcholine on the electrical and secretory activities of frog pituitary melanotrophs.

The activity of melanotroph cells of the amphibian pars intermedia is regulated by multiple factors including classical neurotransmitters and neuropeptides. In this study, we have examined the possible involvement of acetylcholine (ACh) in the regulation of electrical and secretory activities of frog pituitary melanotrophs. Electrophysiological recordings were conducted on cultured cells by using the patch-clamp technique in the whole-cell configuration. In parallel, alpha-MSH release from acutely dispersed pars intermedia cells was studied by means of the perifusion technique. In all cells tested in the current-clamp mode, superfusion with ACh (10(-6) M) gave rise to a depolarization associated with an enhanced frequency of action potentials. Administration of ACh (10(-6) M) to perifused cells also induced stimulation of alpha-MSH release. These results indicate that the neurotransmitter ACh exerts a direct stimulatory effect on pituitary melanotrophs. The action of ACh on electrical and secretory activities was mimicked by muscarine (10(-5) M), while ACh-induced alpha-MSH secretion was completely abolished by the muscarinic antagonist atropine (10(-6) M). The depolarizing effect of muscarine was suppressed by the specific M1 muscarinic antagonist pirenzepine (10(-5) M), indicating the existence of a M1 subtype muscarinic receptor in frog pars intermedia cells. In addition, using a monoclonal antibody against calf muscarinic receptors, we have visualized, by the immunofluorescence technique, the presence of muscarinic receptor-like immunoreactivity in cultured intermediate lobe cells. Electrophysiological recordings showed that nicotine (10(-5) M) induces membrane depolarization associated with an increase of the frequency of action potentials.(ABSTRACT TRUNCATED AT 250 WORDS)

Secretion of MMP-2 and MMP-9 induced by VEGF autocrine loop correlates with clinical features in childhood acute lymphoblastic leukemia

In children with acute lymphoblastic leukemia (ALL), leukemic cells express several members of the VEGF family and the three VEGF receptors which, via an autocrine loop are responsible for secretion of MMP-2/-9. MMP activity and the presence of elements of the autocrine loop are correlated with clinical and prognostic parameters as follows: i) high basal MMP-9 activity with tumoral syndrome, ii) MMP-2 activity with treatment failure, iii) VEGFR-1/-3 co-expression with high hemoglobin level and iv) expression of the VEGF-A 121 isoform and favorable response to treatment. These data implicate autocrine VEGF-induced secretion of MMP-2/-9 in the physiopathology of childhood ALL.