Mediation of cholino-piperine like receptors by extracts of Piper nigrum induces melanin dispersion in Rana tigerina tadpole melanophores

Aim: The present study was carried out to determine the effects of lyophilized dried fruit extracts of Piper nigrum and pure piperine on the tadpole melanophores of frog Rana tigerina which offer excellent in vitro opportunities for studying the effects of pharmacological and pharmaceutical agents. The nature of specific cellular receptors present on the neuro-melanophore junction and their involvement in pigmentary responses has been explored.

Material: Effects of lyophilized extracts of P. nigrum and pure piperine were studied on the isolated tail melanophores of tadpoles of the frog R. tigerina as per the modified method.

Results: The extract of P. nigrum and its active ingredient piperine caused significant melanin dispersal responses leading to darkening of the tail melanophores, which were completely antagonized by atropine and hyoscine. These per se melanin dispersal effects were also found to be markedly potentiated by neostigmine an anticholinesterase agent.

Conclusion: It appears that the melanin dispersal effects of the extracts of P. nigrum and pure piperine leading to skin darkening are mediated by cholinergic muscarinic or piperine-like receptors having similar properties.     

Basic properties of beta adrenergic receptors mediating melanosome dispersion of melanophores in a cyprinid fish, Zacco temmincki

In melanophores of a cyprinid fish, Zacco temmincki, receptor mechanisms of melanosome dispersion induced by catecholamines were examined. While possessing a melanosome-aggregating action in higher concentrations, isoproterenol and epinephrine in lower concentrations acted to disperse melanosomes. Norepinephrine, epinine and dopamine altered their action from melanosome aggregation to melanosome dispersion after alpha adrenergic blockade. The catecholamine-induced melanosome dispersion was inhibited by beta adrenergic blocking agents. Mediation of dispersion is regulated through beta adrenergic receptors. The beta adrenergic responses were unaffected by mersalyl, a sulfhydryl inhibitor. A prospective substance acting in dispersing melanosomes appears to be adrenaline, but not noradrenaline.     

Subtypes of beta adrenergic receptors mediating pigment dispersion in chromatophores of the medaka, Oryzias latipes

Actions of the adrenergic beta-2 agonists, salbutamol and terbutaline, and the beta-1 antagonists, metoprolol and atenolol, were examined on denervated melanophores and leucophores of a teleost, Oryzias latipes. Beta-2 agonists depressed the pigment-aggregation response of melanophores to norepinephrine, while beta-1 antagonists inhibited the dispersion response of leucophores to isoproterenol but not the melanophore response. These findings suggest that adrenergic receptors mediating pigment dispersion in melanophores are beta-2 and those of leucophores are beta-1. The possible relations between receptor mechanisms and the responses of chromatophores are discussed.     

Novel receptors for melatonin that mediate pigment dispersion are present in some melanophores of the pencil fish (Nannostomus)

1. Comparing the daytime and the night-time pigmentary patterns of the skin of the pencil fish, Nannostomus beckfordi, we noticed that specific regions of dark spots that were part of the night-time pattern became pale during the day.2. Microscopic observations revealed that melanosomes in the melanophores in those regions were aggregated during the day but became dispersed at night.3. These melanophores responded to melatonin by dispersal of melanosomes while the cells on other parts of the body responded to melatonin by aggregation of the pigment in the normal way.4. The melanophores that responded to melatonin by pigment dispersion responded normally to other hormones and neurotransmitters, as did those on other parts of the skin.5. The results indicate that, in addition to the known melatonin receptor that mediates the aggregation of melanosomes, there also exists an unusual receptor which mediates the dispersion of pigment in melanophores. We have tentatively designated this receptor the ‘beta-melatonin receptor’.     

Implication of alpha-2 adrenergic post-synaptic receptors in the central catecholaminergic stimulation of the corticotropic axis in rats

We have recently assigned a major stimulatory role to the brain catecholamines (CA) via alpha 1 and beta receptors on CRH-ACTH secretion, e.g. in the physiological response to stress. In the present study, we explored the possible participation in this regulation of post-synaptic alpha 2 receptors in free moving rats, one week after CA denervation of the hypothalamus by bilateral neurotoxic lesions of the noradrenergic ascending brain stem bundles (NAB). Intracerebroventricular (i.c.v.) injection of clonidine (alpha 2 agonist; 1 nmol) induced a 3 fold rise of ACTH release (measured by RIA) above vehicle (PBS) injected controls (p less than 0.001). This stimulatory effect was completely reversed by an i.c.v. pretreatment with the alpha 2 antagonist idazoxan (10 nmol; without action by itself), whereas it was only slightly affected by an i.c.v. pretreatment with a combination of an alpha 1 and beta blocker (prazosin + propranolol; 5/5 nmol; p greater than 0.1). The results strongly suggest the participation of alpha 2 post-synaptic receptors in the central catecholaminergic activation of ACTH secretion.     

A subtype of adenosine receptors mediating pigment dispersion in leucophores of the medaka: evidence for an A2-receptor

1. Adenosine and its derivatives induced dispersion of leucosomes in leucophores of the medaka, Oryzias latipes. 2. Among the purines used, 5'-N-ethylcarboxiamideadenosine was the most effective and its potency was far greater than that of adenosine, N6-L-phenylisopropyladenosine and N6-cyclohexyladenosine. 3. Methylxanthines inhibited the purine action competitively, but beta adrenergic antagonists and dipyridamole did not. 4. Beta adrenergic agonists and forskolin synergistically augmented the purine action, while Li+ blocked it competitively. 5. The results suggest that medaka leucophores possess A2 adenosine receptors on the cell membranes, the stimulation of which induces leucosome-dispersion response by increasing the cellular level of cyclic AMP through activation of adenylate cyclase activity.     

Prostaglandin E2 synthesis elicited by adrenergic stimuli in guinea pig trachea is mediated primarily via activation of beta 2 adrenergic receptors.

Prostaglandin (PG) E2 synthesis elicited by adrenergic agonists in the guinea pig trachea has been shown to be mediated via activation of beta-adrenergic receptors. The purpose of this study was to examine arachidonic acid (AA) metabolism and to characterize the subtype of beta receptor involved in PG synthesis. [14C]AA was incubated with guinea pig tracheal rings, and the radiolabelled products were extracted from the medium. Thin layer chromatographic analysis and radioimmunoassay of the extract showed that [14C]AA was incorporated into guinea pig tracheal rings and metabolized mainly into radiolabeled and immunoreactive PGE2 (iPGE2) and smaller amounts into PGF2 alpha. Trace amounts of PGD2, TxB2 and 6-keto-PGF1 alpha but not LTB4 or LTC4 were detected by enzyme immunoassay. Incubation of guinea pig tracheal rings for 10 min with isoproterenol or salbutamol resulted in a significant increase in PGE2 synthesis (optimum concentration 0.1 microM for both compounds). In contrast, dobutamine, BRL 37344, BRL 28410, norepinephrine, phenylephrine, and xylazine (up to 1 microM) did not significantly increase PGE2 production. Isoproterenol-induced iPGE2 production was inhibited by the selective beta 2 receptor antagonist butoxamine (0.1-1.0 microM) and somewhat reduced by the beta 1 receptor antagonist practolol (1 microM). The increase in PGE2 synthesis was diminished with increasing concentrations of isoproterenol (0.5-5.0 microM) or salbutamol (0.5-1.0 microM); but it was reversed by pretreatment of tracheal rings with the protein synthesis inhibitors cycloheximide (0.9 microM) and actinomycin D (2 microM) but not by phenylisopropyl adenosine (0.1-1.0 microM), an inhibitor of adenylyl cyclase. These data suggest that isoproterenol-induced iPGE2 synthesis is primarily via activation of a beta 2 adrenergic receptor. Failure to enhance iPGE2 synthesis by a high concentration of isoproterenol is likely to be due to an induction of new inhibitory protein synthesis.     

Involvement of the protein kinase pathway in melanin synthesis by chick retinal pigment epithelial cells

Protein kinases are involved in a variety of cellular functions and cell proliferation in eyes. We have explored the involvement of protein kinase C (PKC) in cell proliferation and melanin synthesis by chick retinal pigment epithelial (RPE) cells in vitro. This was achieved by incubation of confluent RPE cells with known inhibitors of protein kinase, H-7, W-7, H-8, and staurosporine. Chick RPE cells were cultured in the presence or absence of the protein kinase inhibitors for a 10-day period. Effects of the inhibitors on cell proliferation and melanin synthesis, as an indication of cell differentiation, were assessed by counting the number of surviving cells and by measuring the melanin content in the cells, respectively. H-7, W-7, and staurosporine inhibited cell proliferation and increased melanin synthesis in a concentration-dependent manner during culture; however, H-8 did not produce these cellular effects. These findings indicate that PKC and calcium/calmodulin-dependent kinase pathways are involved in the proliferation and differentiation of chick RPE cells.     

Protection against alloxan-induced diabetes in mice by stimulation of alpha 2-adrenergic receptors

A single intravenous injection of alloxan in mice induced hyperglycemia in a dose dependent fashion. This diabetogenic action of alloxan was prevented by a single intraperitoneal injection of the alpha 2-adrenergic agonists, i.e. oxymetazoline, clonidine or epinephrine 40 min prior to the injection of alloxan. The alpha 1-adrenergic agonists, i.e. methoxamine and phenylephrine, and a beta-adrenergic agonist, isoproterenol, failed to prevent the diabetogenic action of alloxan. The inhibitory effect of clonidine on alloxan-induced diabetes was antagonized by yohimbine or phentolamine, but not by prazosin. Although alpha 2-adrenergic agonists caused a transient hyperglycemia at the time of alloxan administration (40 min after the administration of alpha 2-adrenergic agonists), the plasma glucose level at the time of alloxan injection did not correlate with the anti-diabetogenic effect of alpha 2-adrenergic agents. These results clearly demonstrate that the alpha 2-adrenergic mechanism which inhibits insulin release from pancreatic B cells prevented the diabetogenic action of alloxan in mice.     

Distribution of the beta-2 adrenergic receptor messenger RNA in the rat brain by in situ hybridization histochemistry: Effects of chronic reserpine treatment

We studied the distribution of the rat brain beta-2 adrenergic receptor (AR) mRNA, and the effects of monoamine depletions by chronic reserpine treatment using in situ hybridization histochemistry. In the control group, high level signals of beta-2 AR mRNA were observed in the parietal, frontal and piriform cortices, the medial septal nuclei, the olfactory tubercle, and the midbrain. Moderate signals were found in the striatum, the retrosplenial cortex, the hippocampus, and the thalamic nuclei. After chronic reserpine treatment, beta-2 AR mRNA levels were increased in many brain regions. The large increases were seen in the hippocampus, all thalamic nuclei, the amygdaloid nuclei, and the midbrain, followed by the striatum and the occipital cortex. The receptor up-regulation resulting from chronic monoamine depletion may be due to these increases in beta-2 AR mRNA, indicating that this up-regulation may be caused by increased receptor production rather than decreased receptor degradation.     

Regulation of Ca2+ influx in myocardial cells by beta adrenergic receptors,cyclic nucleotides,and phosphorylation

Calcium channels in the heart play a major role in cardiac function. These channels are modulated in a variety of ways, including protein phosphorylation. Cyclic AMP-mediated phosphorylation is the best understood phosphorylation mechanism which regulates calcium influx into cardiac cells. Binding of an agonist (e.g., a catecholamine) to the appropriate receptor stimulates production of cyclic AMP by adenylate cyclase. The cyclic AMP may subsequently bind to and activate a cyclic AMP-dependent protein kinase, which then can phosphorylate a number of substrates, including the calcium channel (or a closely-associated regulatory protein). This results in stimulation of the calcium channels, greater calcium influx, and increased contractility. The cyclic AMP system is not the only protein kinase system in the heart. Thus, the possibility exists that other protein kinases may also regulate the calcium channels and, hence, cardiac function. Recent evidence suggests that cyclic GMP-mediated phosphorylation may play a role opposite to cyclic AMP-mediated phosphorylation, i.e., inhibition of the calcium current rather than stimulation. Other recent evidence also suggests that a calcium/calmodulin-dependent protein kinase and calcium/phospholipid-dependent protein kinase (protein kinase C) may also regulate the myocardial calcium channels. Thus, protein phosphorylation may be a general mechanism whereby calcium channels and cardiac function are modulated under a variety of conditions.     

Vasopressin V1 receptors contribute to hemodynamic and sympathoinhibitory responses evoked by stimulation of adenosine A2a receptors in NTS

Activation of adenosine A2a receptors in the nucleus of the solitary tract (NTS) decreases mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA), whereas increases in preganglionic adrenal sympathetic nerve activity (pre-ASNA) occur, a pattern similar to that observed during hypotensive hemorrhage. Central vasopressin V1 receptors may contribute to posthemorrhagic hypotension and bradycardia. Both V1 and A2a receptors are densely expressed in the NTS, and both of these receptors are involved in cardiovascular control; thus they may interact. The responses elicited by NTS A2a receptors are mediated mostly via nonglutamatergic mechanisms, possibly via release of vasopressin. Therefore, we investigated whether blockade of NTS V1 receptors alters the autonomic response patterns evoked by stimulation of NTS A2a receptors (CGS-21680, 20 pmol/50 nl) in alpha-chloralose-urethane anesthetized male Sprague-Dawley rats. In addition, we compared the regional sympathetic responses to microinjections of vasopressin (0.1-100 ng/50 nl) into the NTS. Blockade of V1 receptors reversed the normal decreases in MAP into increases (-95.6 +/- 28.3 vs. 51.4 +/- 15.7 integralDelta%), virtually abolished the decreases in HR (-258.3 +/- 54.0 vs. 18.9 +/- 57.8 integralDeltabeats/min) and RSNA (-239.3 +/- 47.4 vs. 15.9 +/- 36.1 integralDelta%), and did not affect the increases in pre-ASNA (279.7 +/- 48.3 vs. 233.1 +/- 54.1 integralDelta%) evoked by A2a receptor stimulation. The responses partially returned toward normal values approximately 90 min after the blockade. Microinjections of vasopressin into the NTS evoked dose-dependent decreases in HR and RSNA and variable MAP and pre-ASNA responses with a tendency toward increases. We conclude that the decreases in MAP, HR, and RSNA in response to NTS A2a receptor stimulation may be mediated via release of vasopressin from neural terminals in the NTS. The differential effects of NTS V1 and A2a receptors on RSNA versus pre-ASNA support the hypothesis that these receptor subtypes are differentially located/expressed on NTS neurons/neural terminals controlling different sympathetic outputs.     

Neuronal responses are differentially affected by the polarity of tectal DC stimulation in the toad Bufo bufo

Male toads were tested behaviourally for their prey catching responses to worm-like stimuli before being prepared for visual unit and slow potential shift (SPS) recording from the optic tectum. The neuronal responses of toads to a prey-like visual stimulus reflected their motivational tendency prior to operations. One second of DC stimulation to the tectum was followed by an SPS of reversed polarity during which time a visual prey-like stimulus was presented. A negative SPS following positive DC stimulation was associated with enhanced neuronal responses to a visual stimulus. The positive SPS that followed negative stimulation was associated with a decline in neural responses below background when a visual stimulus was additionally given. The SPS was largely a result of DC stimulation that interacted with the motivational tendency to produce enhanced neuronal responses, while the potential was negative and vice versa.     

Involvement of both a Zn2+ site and an anionic binding site in the selective inhibition of a Zn2+-glycerophosphocholine cholinephosphodiesterase by thiols and tellurites

Inhibition of a Zn²⁺-glycerophosphocholine cholinephosphodiesterase by thiols or tellurites were examined mechanistically. Inactivation of the phosphodiesterase by thio-carboxylates, which was due to the removal of Zn²⁺ in the catalytic site, was enhanced by introduction of an amino group in the structure of thiols, suggesting the presence of an anionic site adjacent to a Zn²⁺ site. In support of the suggestion, it was found that thiols, associable with both a Zn²⁺ site and an anionic site, were more potent reversible inhibitors; dimethylaminoethanethiol (Ki, 17 M), diethylaminoethanethiol (Ki, 1.2 M) and thiocholine (Ki, 2.6 M). Meanwhile, the inhibition of the phosphodiesterase by tellurites is ascribed to the binding of tellurite anions to a Zn²⁺ site, based on the protective action of tellurite anions against the inactivation of the enzyme by EDTA. Moreover, the inhibition of the phosphodiesterase by tellurites was prevented by phosphate ions, which expressed the protective effect against EDTA inactivation. In further support, it was observed that gellurite and thiocholine appeared to interact with active site in an additive manner, in contrast to a synergistic action between tellurites and quaternary ammonium compounds such as acetylcholine or choline.     

Thyroid hormone receptors and stimulation of angiotensinogen production in HepG2 cells

Summary Binding characteristics and effects of 3,5,-3′-triiodo-l-thyronine (T₃) on angiotensinogen production in HepG₂ were studied in serum-free medium. Binding was performed on intact cells and on partially purified isolated nuclei using [¹²⁵I]T₃. Scatchard plots revealed one class of high affinity binding sites with a K_d of approximately 80 pmol/liter. Calculation of maximum binding showed that HepG₂ possess approximately 1000 binding sites per cell. Unlabeled T₃ and T₄ competed for binding sites on intact HepG₂ with 50% inhibition of [¹²⁵I]T₃ binding at approximately 3.0 and 38.0 pmol/liter, respectively. The HepG₂ showed a dose-dependent increase in angiotensinogen production in serum-free medium which was maximal at 10⁻⁵ mol/liter (two-fold increase/10⁶ cells/24 h) and had an EC₅₀ of approximately 5.0×10⁻⁸ mol/liter. T₃ also produced after 24 h a dose-dependent increase in DNA highly correlated with T₃ applied (r=0.88,P<0.01). In conclusion, this study shows that HepG₂ possess specific high affinity binding sites for T₃ and that T₃ stimulates angiotensinogen production and DNA synthesis in these cells. Dr. Darby is supported by INSERM (France)/NH and MRC (Australia) exchange fellowship.     

Pharmacological evidence for the stimulation of NADPH oxidase by P2X7 receptors in mouse submandibular glands

ATP in the 100 μM-1 mM concentration range provoked a calcium-independent increase of the oxidation of dichlorodihydrofluorescein (DCFH) to dichlorofluorescein (DCF) by mouse submandibular cells. 3′-O-(4-benzoyl)benzoyl adenosine 5′-triphosphate (BzATP), a P2X₇ agonist, but not a muscarinic or an adrenergic agonist, reproduced the effect of ATP. The inhibition of phospholipase C by U73122 or the potentiation of P2X₄ receptor activation with ivermectin did not modify the response to ATP. ATP did not increase the oxidation of DCFH in cells isolated from submandibular glands of P2X₇ knockout mice or in cells pretreated with a P2X₇ antagonist. The inhibition of protein kinase C or of mitogen-activated protein kinase (MAP kinase) or of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase blocked the oxidation of DCFH without affecting the increase of the intracellular concentration of calcium or the uptake of ethidium bromide in response to extracellular ATP. From these results it is concluded that the activation of the P2X₇ receptors from submandibular glands triggers an intracellular signalling cascade involving protein kinase C and MAP kinase leading to the stimulation of NADPH oxidase and the subsequent generation of reactive oxygen species.     

Dissociation of changes in apparent myofibrillar Ca2+ sensitivity and twitch relaxation induced by adrenergic and cholinergic stimulation in isolated ferret cardiac muscle

In isolated, aequorin-injected ferret cardiac muscle we measured the apparent myofilament Ca2+ sensitivity and its relationship to twitch relaxation time in the presence of autonomic perturbations. The Ca2+-tension relation was determined from the peak aequorin luminescence and peak twitch tension measured in muscles across a broad range of bathing [Ca2+] in the presence and absence of acetylcholine (ACh) (1 microM) or isoproterenol (ISN) (1 microM), or both drugs. ACh shifted the relationship of peak tension to (peak) aequorin light leftward, which suggests an increase in myofilament Ca2+ sensitivity, but it did not alter relaxation, which was measured as the time for peak tension to decay by 50% (t 1/2 R). ISN produced its previously documented effects, i.e., a rightward shift of the relationship of peak tension to peak aequorin light and a decrease in t1/2R. ACh abolished the ISN effect on the peak tension-aequorin light relationship but did not reverse the effect of ISN to decrease t1/2R. The effects of ACh and ISN of modulating the apparent myofilament Ca2+ sensitivity in intact muscles, corroborate findings of previous studies in isolated myofibrillar preparations. However, these perturbations of myofilament Ca2+ sensitivity in the intact muscle do not relate to twitch relaxation, measured as t1/2R, since (a) ACh affects the former but not the later and (b) the effect of ISN on the Ca2+-tension relationship is abolished by ACh, while the relaxant effect persists.     

α-adrenergic inhibition of cyclic AMP accumulation in hamster adipocytes similarity of receptor with α-2 adrenergic receptors

The present communication shows the effects of several α-adrenergic agonists and antagonists on cyclic AMP levels in hamster epididymal adipocytes. In response to ACTH (30 mU/ml) in combination with 1-methyl-3-isobutylxanthine (0.10 mM) or adenosine deaminase (1.0 μg/ml), cyclic AMP levels increased to a maximum by 10 min and this level was maintained for another 20 min. Elevated cyclic AMP levels were partially suppressed by the α-adrenergic agents clonidine, methoxamine, methyl norepinephrine and phenylephrine. The lowest effective concentration of each of these agonists required to suppress cyclic AMP levels was 10 nM clonidine; 3 μM methoxamine; 10 μM methyl norepinephrine; 10 μM phenylephrine. Clonidine and methoxamine suppressed cyclic AMP levels by nearly 65% while phenylephrine and methyl norepinephrine caused only a 30% decline. Studies of the relative potencies of α-adrenergic blocking drugs on prevention of the inhibitory effect of clonidine on cyclic AMP levels disclosed that phentolamine and yohimbine were more potent blockers of clonidine action than phenoxybenzamine and prazosin. The rank order of potencies of agonists at causing suppression of cyclic AMP levels and the rank order of potencies of antagonists of clonidine action suggest similarity of the α-adrenergic receptors present on hamster adipocytes, which affect cyclic AMP accumulation to α-2 adrenergic receptors.