Pituitary adenylate cyclase-activating polypeptide (PACAP38) modulates lymphocyte and macrophage functions: stimulation of adherence and opposite effect on mobility

The effects of pituitary adenylate cyclase-activating polypeptide (PACAP38) in a concentration range from 10⁻¹³ to 10⁻⁶ M were studied, in vitro, on two functions of peritoneal rat lymphocytes and macrophages: adherence and mobility (spontaneous and chemotaxis). The results show that PACAP38 raised the adherence of the two cell types, increased the mobility of macrophages and decreased the mobility of lymphocytes. The maximal effects were observed at 10⁻¹⁰ M in macrophages and at 10⁻⁹ M in lymphocytes. Moreover, incubation with increasing concentrations of phorbol myristate acetate (PMA), a protein kinase C (PKC) activator, resulted in a progressive enhancement of adherence and chemotaxis of both macrophages and lymphocytes. In contrast, retinal, a PKC inhibitor, significantly decreased these capacities. Incubation of macrophages with both PMA and PACAP38 did not have a synergistic effect on chemotaxis and adherence whereas, with lymphocytes, adherence was increased and chemotaxis was partially decreased. On the other hand, incubation with forskolin (an enhancer of intracellular cyclic AMP [cAMP]levels) caused inhibition and stimulation of chemotaxis and adherence, respectively, in both cell types. PACAP38 prevented the inhibitory effect of forskolin on chemotaxis of macrophages but not of lymphocytes, whereas the simultaneous presence of PACAP38 and forskolin was synergistic for adherence of both peritoneal cells. In addition, PACAP38 was chemoattractant for macrophages but not for lymphocytes. Furthermore, a VIP receptor antagonist was able to partially reverse the modulatory effects of PACAP38 on lymphocytes, but not on macrophages. These data suggest that PACAP38 exerts its action through the binding to type I PACAP receptors and PKC activation in macrophages and through the elevation of intracellular cAMP levels by binding to type II PACAP receptors in lymphocytes. The present work reveals an additional link between neuropeptides and the immune system and suggests that the peptide PACAP modulates the immunological function of macrophages and lymphocytes.     

Vasoconstrictor effect of the Ca agonist Bay K 8644 on human cerebral arteries

The effects of Bay K 8644 on the reactivity and ⁴⁵Ca²⁺ uptake in segments from human cerebral arteries were studied. Bay K 8644 induced concentration-dependent contractions up to 10⁻⁶ M; 10⁻⁵ M produced a reduction of the maximal response. The Ca²⁺ agonist elicited these contractions by itself, and no previous depolarization was needed. The response to Bay K 8644 was antagonized competitively by nifedipine (5 × 10⁻⁸ and 10⁻⁷ M, πA₂ value of 8.17) and non-competitively by verapamil (10⁻⁶, 5 × 10⁻⁶ and 10⁻⁵ M). The contraction induced by 10⁻⁷ M Bay K 8644 was inhibited by a Ca²⁺-free medium containing 1 mM EGTA. The subsequent cumulative Ca²⁺ addition, caused concentration-dependent contractions up to 2.5 mM Ca²⁺, which were reduced by nifedipine (10⁻⁸ and 10⁻⁷ M) or verapamil (5 × 10⁻⁶ and 10⁻⁵ M). When the EGTA concentration in the Ca²⁺-free solution was reduced to 0.1 mM, contractions induced by Ca²⁺ up to 5 mM, including 0 Ca²⁺, were increased with respect to those obtained in the presence of 1 mM EGTA. Basal ⁴⁵Ca²⁺ uptake was not modified with Bay K 8644 (10⁻⁶ M) or nifedipine (10⁻⁶ M). K⁺ (25 and 50 mM) produced an increase on ⁴⁵Ca²⁺ uptake, which was potentiated by Bay K 8644 (10⁻⁶ M) and antagonized by nifedipine (10⁻⁶ M); this latter agent reduced the potentiation elicited by the Ca²⁺ agonist. These results suggest that contractions caused by Bay K 8644 in human cerebral arteries are produced by the opening of the voltage-dependent Ca²⁺ channels present in vascular smooth muscle cells, which appear to be preactivated in a basal situation, facilitating Ca²⁺ influx. Nevertheless, Bay K 8644 needed a previous cell depolarization to produce an increase in ⁴⁵Ca²⁺ uptake.     

Neurotensin and neuromedin N elevate the cytosolic calcium concentration via transiently appearing neurotensin binding sites in cultured rat cortex cells

Through assessment of the changes in the intracellular free-calcium concentration ([Ca²⁺]_i), which was measured using the calcium sensitive dye, fura-2, the character of the neurotensin (NT) binding sites which appeared transiently during the early ontogenetic stage in the rat cerebral cortex was analyzed in primary cultures of cerebral cortex cells from neonatal rats. NT (1–1000 nM) elevated [Ca²⁺]_i of the cells even when extracellular calcium was chelated with 1 mM ethylene glycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA). These findings suggest that the transiently appearing NT-binding sites in the cortex are receptors for NT and that some of the NT-induced increase in [Ca²⁺]_i is due to mobilization from the intracellular calcium store. Further application of NT after 10 min washing caused an increase in [Ca²⁺]_i again. This is in contrast to the findings for cortical slices from adult rats and mRNA-injected oocytes; desensitization due to NT was of long duration and further application of NT failed to activate the neurons which had responded the first time to NT. These facts suggest that the character of the NT-binding sites in the cerebral cortex differs between neonatal and adult rats. In addition, we showed that neuromedin N had a similar property to NT as to mobilization of [Ca²⁺]_i and acted only on NT-responsive cells, suggesting the interaction between NT and neuromedin N at the postsynaptic level via the same receptor.     

Prolonged enhancement of synaptic transmission in area CA1 of rat hippocampal slices induced by NaF/AlCl3 does not require NMDA receptor activation but is suppressed by inhibitors of phosphoinositide-mediated signalling pathways

The processes underlying the action of AlF₄⁻ (10 mM NaF/10 μM AlCl₃) in inducing long-lasting enhancement of synaptic transmission in area CA1 of rat hippocampal slices have been investigated. Exposure of hippocampal slices to AlF₄⁻ for 10 min caused population EPSP slope to rise by approximately 50% within 60 min of washing off the NaF/AlCl₃ saline. This effect was not inhibited either by APV (50 μM), or by temporary interruption of the delivery of test stimuli during and for up to 20 min after application of the AlF₄⁻-containing medium. However, pretreatment of preparations with either thapsigargin (1 μM) or staurosporine (1 μM), or ommission of Ca²⁺ from the AlF₄⁻-containing saline (no addition of EGTA) prevented the potentiating action of NaF/AlCl₃. We conclude that the potentiating effect of AlF₄⁻ is via a G-protein linked to phosphoinositide turnover, and that both arms of this signalling pathway are necessary for potentiation to occur. Ca²⁺ influx is also a requirement, but does not occur through the NMDA receptor.     

Increase of calmodulin-stimulated striatal particulate phosphorylation response in chronic haloperidol-treated rats

Calcium- and calmodulin-dependent protein kinase and phosphatase activities were studied in rat striatal particulate preparations. The effect of Ca²⁺ (0.1–0.5 mM) on phosphorylation was completely abolished in the preparation which had been washed 3 times with a buffer containing ethylene glycol bis-(β-aminoethyl ether)-N,N′-tetraacetic acid (EGTA, 1.2 mM). Ca²⁺-stimulated phosphorylation was restored in a dose-dependent manner after calmodulin (1 μg) was added to the assays. Ca²⁺ and calmodulin promoted the phosphate incorporation into two major striatal protein bands with estimated M_r at 52 and 40 kdaltons. The presence of phosphatase in the EGTA-pretreated preparations was negligible. Chronic treatment in rats with haloperidol (1 mg/kg, 20 days) produced a significant decrease in the Ca²⁺-independent phosphorylation but an increase in the extent of Ca²⁺ and calmodulin-dependent phosphorylation in the striatum. The chronic haloperidol treatment did not alter the striatal [¹²⁵I]calmodulin binding curve. In vitro, haloperidol (even at 10⁻⁴ M) had no effect on calmodulin-dependent phosphorylation. Haloperidol (10⁻⁴ M) did not reduce the number but decreased the rate of [¹²⁵I]calmodulin binding to the striatal particulates. These data suggest that the link between dopamine receptors and calmodulin-dependent enzyme is dissociated in vitro. On the other hand, the potential sensitivity of calmodulin-dependent protein kinase in the chronic haloperidol-treated rats correlated with the supersensitive dopamine receptor responses occurred in these rats. Therefore, calmodulin-dependent protein kinase may biochemically regulate dopamine receptor function in the striatum in vivo.     

Neurotensin — macrophage interaction: Specific binding and augmentation of phagocytosis

Neurotensin (NT) was found to bind to thioglycollate-elicited mouse peritoneal macrophages and to modulate their phagocytic capability. A Scatchard analysis of the binding curve of [³H]NT suggests the presence of two subclasses of binding sites having a.−a K_D of 0.9 nM (4800 sites per cell) and b.−a K_D of 28 nM (33500 sites per cell). NT as well as two of its partial sequences, NT(8–13) and NT(6–13) competed with [³H]NT for its binding whereas NT(1–10) was rather ineffective. [³H]NT was also competitively displaced by tuftsin, substance P (SP) and by SP (1–4). The K_I values estimated for all the above competitive inhibitors of [³H]NT binding (except for NT) suggest interaction with the relatively low affinity sites.NT exerts a biphasic effect on the phagocytic response of macrophages. At a concentration range of 10⁻¹⁴–10⁻⁹ M NT had a dose dependent augmenting effect on the phagocytic response (up to 2 fold), further increase in concentration (>10⁻⁹M) of NT resulted in a gradual decrease of the augmented response which disappears at 10⁻⁷ M NT. NT(8–13), NT(6–13) as well as NT(1–10) augment the phagocytic response of macrophages. However the maximal effect with these peptides was attained at about 10⁻⁷ M and stayed at the same level at concentrations up to 10⁻⁵ M. The phagocytosis augmenting dose-response curves of these peptides resembled that of tuftsin and SP, two unrelated peptides.It is suggested that NT-phagocyte interaction may be of relevance in the regulation of the functions of phagocytic cells.     

Ionic currents on type-I cells of the rabbit carotid body measured by voltage-clamp experiments and the effect of hypoxia

Type-I cells (from rabbit embryos) in primary culture were studied in voltage-clamp experiments using the whole cell arrangement of the patch-clamp technique. With a pipette solution containing 130 mM K⁺ and 3 mM Mg-ATP, large outward currents were obtained positive to a threshold of about −30 mV by clamping cells from −50 mV to different test pulses (−80 to 50 mV). Negative to −30 mV, the slope conductance was low (outward rectification). The outward currents were blocked by external Cs⁺ (5 mM) and partially blocked by TEA (5 mM) and Co²⁺ (1 mM). The initial part of the outward currents during depolarizing voltage pulses exhibited a transient Ca²⁺ inward component partially superimposed to a Ca²⁺-dependent outward current. Inward currents were further characterized by replacing K⁺ with Cs⁺ in the intra- and extracellular solution in order to minimize the outward component and by using 1.8 mM Ca²⁺ or 10.8 mM Ba²⁺ as charge carrier. Slow-inactivating inward currents were recorded at test potentials ranging from −50 to 40 mV (holding potential −80 mV). The maximal amplitude, measured at 10 mV in the U-shaped I–V curve, amounted to 247 ± 103pA(n = 3). This inward current was insensitive to 3 μM TTX, but blocked by 1 mM Co²⁺ and partially reduced by 10 μM D600 and 3 μM PN 200-110. In contrast to outward currents, the inward currents exhibited a ‘run-down’ within about 10 min. Lowering the pO₂ from the control of 150 Torr (air-gassed medium) to 28 Torr had no apparent effect on inward currents, but depressed reversibly outward currents by 28%. In conclusion, it is suggested that type-I cells possess voltage-activated K⁺ and Ca²⁺ channels which might be essential for chemoreception in the carotid body.     

Activity-evoked extracellular pH shifts in slices of rat dorsal lateral geniculate nucleus

Activity-dependent extracellular pH shifts were studied in slices of the rat dorsal lateral geniculate nucleus (dLGN) using double-barreled pH-sensitive microelectrodes. In 26 mM HCO₃⁻-buffered media, afferent activation (10 Hz, 5 s) elicited an early alkaline shift of 0.04±0.02 pH units associated with a later, slow acid shift of 0.05±0.03 pH units. Extracellular pH shifts in the ventral lateral geniculate nucleus were rare, and limited to acidifications of approximately 0.02 pH units. The alkaline shift in the dLGN increased in the presence of benzolamide (1–2 μM), an extracellular carbonic anhydrase inhibitor. The mean alkaline shift in benzolamide was 0.10±0.05 pH units. In 26 mM HEPES-buffered saline, the alkaline response averaged 0.09±0.03 pH units. The alkaline shifts persisted in 100 μM picrotoxin (PiTX) but were blocked by 25 μM CNQX/50 μM APV. If stimulation intensity was raised in the presence of CNQX/APV, a second alkalinization arose, presumably due to direct activation of dLGN neurons. The direct responses were amplified by benzolamide, and blocked by either 0 Ca²⁺/EGTA, Cd²⁺ or TTX. In 0 Ca²⁺, addition of 500 μM–5 mM Ba²⁺ restored the alkalosis. Alkaline shifts evoked with extracellular Ba²⁺ were larger and faster than those elicited by equimolar Ca²⁺. In summary, synchronous activation in the dLGN results in an extracellular H⁺ sink, via a Ca²⁺-dependent mechanism, similar to activity-dependent alkaline shifts in hippocampus.     

ATP-dependent [H]met-enkephalin uptake by bovine adrenal chromaffin granule membrane

Uptake of [³H]Met-enkephalin by purified chromaffin granule membrane isolated from bovine adrenal medulla was investigated. Addition of a single divalent cation, such as Mg²⁺ or Ca²⁺, did not affect the uptake. The presence of 3 mM ATP in the incubation medium stimulated the uptake to two times that of the control. The same stimulation effect was also found in the presence of ATP plus a single divalent action. The ATP-dependent uptake reached its half-maximal level within 5 min after initiation of the reaction at 25°C, and reached a plateau within 10 min. The apparentK_m for [³H]Met-enkephalin uptake by the chromaffin granule membrane was about 2.3 × 10⁻⁶ M. GTP, UTP, UTP and ADP did not stimulate enkephalin uptake. Several calcium inhibitors such as trifluoperazine, verapamil and 3,4,5-trimethoxybenzoic acid 8-(N,N-diethylamino)octylester (TMB-8) markedly inhibited enkephalin uptake. The inhibition exceeded 80%, although these chemicals inhibited Ca²⁺-ATPase activity in chromaffin granule membrane only 30% under the same conditions. Ethyleneglycol-bis-(β-aminoethylether)N,N′-tetraacetic acid (EGTA), at 3 mM also inhibited uptake about 30%. The results indicate that uptake of Met-enkephalin by chromaffin granule membrane ghosts was driven not only by ATPase activity, but also by some other Ca²⁺-ATP-mediated mechanism(s).     

A new type of Ca channel blocker, NC-1100, inhibits the low- and high-threshold Ca currents in the rat CNS neurons

The effect of a new type of organic Ca²⁺ channel blocker, NC-1100 [(±)-1-(3,4-dimethoxyphenyl)-2-(4-diphenylmethylpiperazinyl)ethanol dihydrochloride], on both low- and high-threshold Ca²⁺ currents was studied in the whole-cell mode of the pyramidal neurons freshly dissociated from rat hippocampal CA1 region under voltage-clamp condition. The NC-1100 reversibly reduced the high-threshold Ca²⁺ current (HVAI_Ca) in a concentration-dependent manner without affecting the current-voltage relationship. The values of half-inhibition (IC₅₀) were 1.3 × 10⁻⁵ and 9.1 × 10⁻⁶M in external solution containing 10 and 2.5 mM Ca²⁺, respectively. The NC-1100 also decreased the low-threshold Ca²⁺ current (LVAI_Ca) in a concentration-dependent manner. The inhibitory potency was augmented by increasing the stimulation frequency and / or decreasing the extracellular Ca²⁺ concentration to a physiological range (2.5 mM). The IC₅₀ value decreased to 7.7 × 10⁻⁷M in external solution containing 2.5 mM Ca²⁺ at a stimulation frequency of 1 Hz. The NC-1100 delayed the reactivation of LVA Ca²⁺ channel and enhanced voltage-dependently the steady-state inactivation, suggesting that this drug bound not only the resting LVA Ca²⁺ channel but also the inactivated one.     

Hyposmotic activation hyperpolarizes outer hair cells of guinea pig cochlea

The electrophysiological responses of isolated guinea pig outer hair cells (OHCs) to hyposmotic activation were studied using the whole-cell patch-clamp technique. The cell swelling by hyposmotic activation hyperpolarized OHCs by 6.6 ± 2.3 mV from the resting membrane potential of −58.5 ± 5.9 mV (n = 48). This hyperpolarization was associated with an outward current ( 97.7 ± 22.2, pA, n = 15). The hyperpolarization was inhibited by 300 μM quinine, 5 mN Ba²⁺ and increasing the extracellular K⁺ to 30 mM from 5 mM. In the absence of extracellular Ca²⁺ (1 mM EGTA), the hyperpolarization during hyposmotic activation was also abolished while the following depolarization was preserved. 50 μM GdCl₃, which is known to block strecch-activated non-specific cation channels, inhibited the hyperpolarization reversibly. 50 μM GdCl₃ also inhibited [Ca²⁺]_i increase during hyposmotic activation as shown by the calcium-sensitive dye fura-2. Simultaneously, the [Ca²⁺]_i increase and the hyperpolarization during hyposmotic activation could be observed using the combined method of whole-cell patch clamp and fura-2 technique. It is concluded that the cell swelling by hyposmotic activation may activate the stretch-activated non-specific cation channels in the OHCs which allow a Ca²⁺ influx. In turn, this [Ca²⁺]_i increase leads to an activation of the Ca²⁺-activated K⁺ channels at the basolateral membrane of OHCs which results finally in a reversible hyperpolarization of OHCs by K⁺ efflux.     

Regulation of Ca-activated nonselective cationic currents in rat pituitary GH3 cells: involvement in L-type Ca current

Ionic currents were investigated by a patch clamp technique in a clonal strain of pituitary (GH₃) cells, using the whole cell configuration with Cs⁺ internal solution. Depolarizing pulses positive to 0 mV from a holding potential of −50 mV activated the voltage-dependent L-type Ca²⁺ current (I_Ca,L) and late outward current. Upon repolarization to the holding potential, a slowly decaying inward tail current was also observed. This inward tail current upon repolarization following a depolarizing pulse was found to be enhanced by Bay K 8644, but blocked by nifedipine or tetrandrine. This current was eliminated by Ba²⁺ replacement of external Ca²⁺ as the charge carrier through Ca²⁺ channels, removal of Ca²⁺ from the bath solution, or buffering intracellular Ca²⁺ with EGTA (10 mM). The reversal potential of inward tail current was approximately −25 mV. When intracellular Cl⁻ was changed, the reversal potential of the Ca²⁺-activated currents was not shifted. Thus, this current is elicited by depolarizing pulses that activate I_Ca,L and allow Ca²⁺ influx, and is referred to as Ca²⁺-activated nonselective cationic current (I_CAN). Without including EGTA in the patch pipette, the slowly decaying inward current underlying the long-lasting depolarizing potential after Ca²⁺ spike was also observed with a hybrid current–voltage protocol. Thus, the present studies clearly indicate that Ca²⁺-activated nonselective cationic channels are expressed in GH₃ cells, and can be elicited by the depolarizing stimuli that lead to the activation of I_Ca,L.     

Pituitary adenylate cyclase-activating polypeptides ( PACAP27 and PACAP38) inhibit the mobility of murine thymocytes and splenic lymphocytes: comparison with VIP and implication of cAMP

In the present study, the effects of PACAP27, PACAP38 and VIP in a concentration range from 10⁻¹³ to 10⁻⁶ M were studied in vitro on the spontaneous and directed mobility of lymphocytes from rat spleen and thymus. The results show that VIP and both PACAPs inhibit significantly and in a similar way the mobility of lymphocytes from thymus and spleen, and the maximal effects were observed at 10⁻⁹ M and 10⁻⁸ M. The three neuropeptides significantly increased cAMP concentrations. Moreover, incubation with increasing PMA concentrations showed a progressive enhancement of chemotaxis of lymphocytes, which was partially prevented by VIP, and both PACAPs. Incubation with forskolin caused decrease in the chemotaxis of thymocytes and splenocytes, and the presence of VIP or PACAP peptides was not synergistic in the inhibitory effect on lymphocyte chemotaxis, suggesting that the three neuropeptides and forskolin mediate their actions by the same intracellular pathway. This study showed the ability of the VIP receptor antagonist (N-Ac-Tyr¹, D-Phe²)-GRF(l-29)-NH₂ to partially reverse the inhibitory effect of both PACAPs and VIP on chemotaxis, suggesting that PACAP receptors are identical or very similar to VIP receptors in both thymocytes and splenocytes. These data suggest that PACAP27 and PACAP38 can be included as two novel immunoregulatory peptides that can modulate cell mobility on central and peripheral lymphoid organs.     

Effect of KB-2796, a new diphenylpiperazine Ca antagonist, on voltage-dependent Ca currents and oxidative metabolism in dissociated mammalian CNS neurons

The effects of KB-2796, 1-[bis(4-fluorophenyl)methyl]-4-(2,3,4-trimethoxybenzyl)piperazine-2HCl, on the low- and high-voltage activated Ca²⁺ currents (LVA and HVA I_Ca, respectively) and on oxidative metabolism were studied in neurons freshly dissociated from rat brain. KB-2796 reduced the peak amplitude of LVA I_Ca in a concentration-dependent manner with a threshold concentration of 10⁻⁷ M when the LVA I_Ca was elicited every 30 s in the external solution with 10 mM Ca²⁺. The concentration for half-maximum inhibition (IC₅₀) was 1.9 × 10⁻⁶M. At 10⁻⁵ M or more of KB-2796, a complete suppression of the LVA I_Ca was observed in the majority of neurons tested. There was no apparent effect on the current-voltage (I-V) relationship and the current kinetics. KB-2796 delayed the reactivation and enhanced the inactivation of the Ca²⁺ channel for LVA I_Ca voltage- and time-dependently, suggesting that KB-2796 preferentially binds to the inactivated Ca²⁺ channel. KB-2796 at a concentration of3.0 × 10⁻⁶M also decreased the peak amplitude of the HVA I_Ca without shifting the I-V relationship. In addition, KB-2796 reduced the oxidative metabolism (the formation of reactive oxygen species) of the neuron in a concentration-dependent manner with a threshold concentration of3 × 10⁻⁶M. It is suggested that the inhibitory action of KB-2796 on the neuronal Ca²⁺ influx and the oxidative metabolism, in combination with a cerebral vasodilatory action, may reduce ischemic brain damage.     

Preferential inhibition of L- and N-type calcium channels in the rat hippocampal neurons by cilnidipine

The effect of a dihydropyridine Ca²⁺ antagonist, cilnidipine, on voltage-dependent Ca²⁺ channels was studied in acutely dissociated rat CA1 pyramidal neurons using the nystatin-perforated patch recording configuration under voltage-clamp conditions. Cilnidipine had no effect on low-voltage-activated (LVA) Ca²⁺ channels at the low concentrations under 10⁻⁶ M. On the other hand, cilnidipine inhibited the high-voltage-activated (HVA) Ca²⁺ current (I_Ca) in a concentration-dependent manner and the inhibition curve showed a step-wise pattern; cilnidipine selectively reduced only L-type HVA I_Ca at the low concentrations under 10⁻⁷ and 10⁻⁶ M cilnidipine blocked not only L- but also N-type HVA I_Ca. At the high concentration over 10⁻⁶ M cilnidipine non-selectively blocked the T-type LVA and P/Q- and R-type HVA Ca²⁺ channels. This is the first report that cilnidipine at lower concentration of 10⁻⁶ M blocks both L- and N-type HVA I_Ca in the hippocampal neurons.     

Effects of intravesicular loading of a Ca2+ chelator and depolymerization of actin fibers on neurotransmitter release in frog motor nerve terminals

Ca²⁺‐induced Ca²⁺ release (CICR) via type‐3 ryanodine receptor enhances neurotransmitter release in frog motor nerve terminals. To test a possible role of synaptic vesicle in CICR, we examined the effects of loading of EGTA, a Ca²⁺ chelator, into synaptic vesicles and depolymerization of actin fibers. Intravesicular EGTA loading via endocytosis inhibited the ryanodine sensitive enhancement of transmitter release induced by tetanic stimulation and the associated rises in intracellular‐free Ca²⁺ ([Ca²⁺]_i: Ca²⁺ transients). Latrunculin A, a depolymerizer of actin fibers, enhanced both spontaneous and stimulation‐induced transmitter release, but inhibited the enhancement of transmitter release elicited by successive tetanic stimulation. The results suggest a possibility that the activation of CICR from mobilized synaptic vesicles caused the enhancement of neurotransmitter release.     

Effects of neurotensin on neurons in the rat central amygdaloid nucleus in vitro

The effects of neurotensin (NT) on neurons in the central amygdaloid nucleus (ACe) were investigated in rat brain slice preparations by adding the peptide to the perfusing medium. Of 115 ACe neurons, 69 cells (60%) showed excitatory responses and 10 cells (9%) showed inhibitory responses to application of NT. The excitatory response to NT was observed in a dose-dependent manner and the threshold concentration was approximately 3 × 10⁻⁹ M. The excitatory effects of NT persisted under blockade of synaptic transmission. The NT fragment neurotensin 8–13 and the NT analogue neuromedin N showed effects similar to those of NT, whereas the NT fragment neurotensin 1–8 had no effect on ACe neurons. Of 43 neurons in the septal nucleus, 8 cells (19%) and 3 cells (7%) showed excitatory and inhibitory responses, respectively, to NT. The results suggest that NT exerts a potent excitatory effect on ACe neurons through a direct action on specific receptors, in which NT may play a role in amygdala-relevant functions.     

Inhibitory effect of regucalcin on Ca-dependent protein kinase activity in rat brain cytosol: involvement of endogenous regucalcin

The effect of regucalcin, a Ca²⁺-binding protein, on Ca²⁺-dependent protein kinase activity in the brain cytosol of rats with different ages (5 and 50 weeks old) was investigated. The addition of calmodulin (10 μg/ml) or dioctanoylglycerol (5 μg/ml) in the enzyme reaction mixture caused a significant increase in protein kinase activity in the presence of CaCl₂ (1 mM), indicating that Ca²⁺ calmodulin or protein kinase C is present in the cytosol. Such an increase was completely prevented by the addition of regucalcin (10⁻⁷ M). Moreover, regucalcin (10⁻⁷ M) significantly inhibited cytosolic protein kinase activity without Ca²⁺/calmodulin or dioctanoylglycerol addition. Meanwhile, the presence of anti-regucalcin monoclonal antibody (10–50 ng/ml) in the enzyme reaction mixture caused a significant elevation of protein kinase activity, suggesting an inhibitory effect of endogenous regucalcin. Brain cytosolic protein kinase activity was significantly elevated by increasing age (50-week-old rats). Also, regucalcin (10⁻⁷ M) significantly decreased protein kinase activity without Ca²⁺ addition in the brain cytosol of aged rats. However, the effect of anti-regucalcin monoclonal antibody (50 ng/ml) in elevating protein kinase activity was not seen in the brain cytosol of aged rats. These results suggest that regucalcin has an inhibitory effect on Ca²⁺-dependent protein kinase activity in rat brain cytosol, and that the effect of endogenous regucalcin may be weakened in the brain cytosol of aged rats.     

Structure-activity studies with car☐y- and amino-terminal fragments of neurotensin on hypothalamic neurons in vitro

The purpose of this study was to determine the structural requirements for the activity of neurotensin (NT_1–13) on preoptic/anterior hypothalamic (POAH) neurons in vitro. Standard explant culture electrophysiological techniques were employed. NT was administered to POAH cultures through the superfusion fluid, or, to the vicinity of individual neurons by pressure ejection (0.5–10 psi) from micropipettes. Computer-generated, peri-event histograms were used to quantitate neuronal responses. Pressure ejection of NT_1–13 (50 pM to 1 μM) consistently produced an excitatory effect on 30 of 42 neurons. The reamaining cells were either inhibited or unaffected. Application of the C-terminal hexapeptide, NT_8–13, but not the N-terminal octapeptide, NT_1–8 (</1 mM), produced an excitatory response in 21 of 30 neurons, but was less potent than NT_1–13. Application of an N-acetylated NT_8–13 fragment (NT_AC8–13) produced a response that was similar to that produced by NT_8–13. The excitatory effects of NT_1–13 were maintained in medium which effectively blocked synaptic transmission (0 mM Ca²⁺/12 mM Mg²⁺ 1 mM EGTA). These data indicate that the C-terminal hexapeptide, but not the N-terminal octapeptide, produces a dose-related, excitatory effect on single neurons in the POAH in vitro. The persistence of these effects in Ca²⁺-free medium supports a postsynaptic site of action for these peptides.     

Release of immunoreactive α-MSH by synaptosome-enriched fractions of homogenates of hypothalami

Immunoreactive α-melanocyte-stimulating hormone (α-MSH) was found to be concentrated in a synaptosome-enriched fraction prepared by differential centrifugation of rat hypothalamic homogenates. The release of the hormone from this preparation was investigated. After incubation, the synaptosomes were isolated by ultrafiltration and α-MSH in the ultrafiltrate was determined by radioimmunoassay. Particle-bound α-MSH, recovered by extraction with acid ethanol, and α-MSH released from the synaptosome preparation, were immunologically similar to synthetic α-MSH and had an accompanying melanotropic activity. Less than 10% of the particle-bound α-MSH was released during incubation in 0.32 M sucrose. However, in the presence of 2 mM Ca²⁺, α-MSH release increased with increasing concentrations (30–150 mM) of K⁺. The stimulatory effect of 60 mM K⁺ was complete within 2 min and was potentiated by increasing Ca²⁺ concentrations over the range of 0 to 2 mM. K⁺-induced release of α-MSH was independent of temperature from 1 to 30°C, and neither glucose (10 mM) nor dopamine (10⁻¹⁰-10⁻² M) had any effect on the release of the peptide. It is concluded that a synaptosome-enriched fraction from the hypothalamus contains a releasable pool of immunoreactive α-MSH that is mobilized by depolarizing concentrations of K⁺ in a Ca²⁺-dependent manner.