Effect of a p38 MAPK inhibitor on FFA-induced hepatic insulin resistance in vivo.

The mechanisms whereby prolonged plasma free fatty acids elevation, as found in obesity, causes hepatic insulin resistance are not fully clarified. We herein investigated whether inhibition of p38 mitogen-activated protein kinase (MAPK) prevented hepatic insulin resistance following prolonged lipid infusion. Chronically cannulated rats were subdivided into one of four intravenous (i.v.) treatments that lasted 48 h: Saline (5.5 µl min(-1)), Intralipid plus heparin (IH, 20% Intralipid+20 U ml(-1) heparin; 5.5 µl min(-1)), IH+p38 MAPK inhibitor (SB239063) and SB239063 alone. During the last 2 h of treatment, a hyperinsulinemic (5 mU kg(-1) min(-1)) euglycemic clamp together with [3-(3)H] glucose methodology was carried out to distinguish hepatic from peripheral insulin sensitivity. We found that SB239063 prevented IH-induced hepatic insulin resistance, but not peripheral insulin resistance. SB239063 also prevented IH-induced phosphorylation of activating transcription factor 2 (ATF2), a marker of p38 MAPK activity, in the liver. Moreover, in another lipid infusion model in mice, SB239063 prevented hepatic but not peripheral insulin resistance caused by 48 h combined ethyloleate plus ethylpalmitate infusion. Our results suggest that inhibition of p38 MAPK may be a useful strategy in alleviating hepatic insulin resistance in obesity-associated disorders.

Gonadotrophin-releasing hormone pulse generator activity in the hypothalamus of the goat.

Pulsatile release of gonadotrophin-releasing hormone (GnRH) is indispensable to maintain normal gonadotrophin secretion. The pulsatile secretion of GnRH is associated with synchronised electrical activity in the mediobasal hypothalamus (i.e. multiple unit activity; MUA), which is considered to reflect the rhythmic oscillations in the activity of the neuronal network that drives pulsatile GnRH secretion. However, the cellular source of this ultradian rhythm in GnRH activity is unknown. Direct input from kisspeptin neurones in the arcuate nucleus (ARC) to GnRH cell bodies in the medial preoptic area or their terminals in the median eminence could be the intrinsic source for driving the GnRH pulse generator. To determine whether kisspeptin signalling could be responsible for producing pulsatile GnRH secretion, we studied goats, measured plasma levels of luteinising hormone (LH) and recorded MUA in the posterior ARC, where the majority of kisspeptin neuronal cell bodies are located. Rhythmic volleys of MUA were found to be accompanied by LH pulses with regular intervals in the ARC, where kisspeptin neuronal cell bodies were found. Exogenous administration of kisspeptin stimulated a sustained increase in LH secretion, without influencing MUA, suggesting that the GnRH pulse generator, as reflected by MUA, originated from outside of the network of GnRH neurones, and could plausibly reflect the pacemaker activity of kisspeptin neurones, whose projections reach the median eminence where GnRH fibres project. These observations suggest that the kisspeptin neurones in the ARC may be the intrinsic source of the GnRH pulse generator.

Modulation of gonadotrophin-releasing hormone pulse generator activity by the pheromone in small ruminants.

In small ruminants, such as goats and sheep, a primer pheromone produced by males induces an out-of-seasonal ovulation in anoestrous females, a phenomenon known as the male effect. The male effect is unique in that an external chemical stimulus can immediately modulate the activity of the hypothalamic gonadotrophin-releasing hormone (GnRH) pulse generator. We have established a monitoring method of the GnRH pulse generator activity in Shiba goat. Using this method as a sensitive bioassay to assess the male effect pheromone activity, we have shown that the male effect pheromone is synthesised in an androgen-dependent manner in the sebaceous glands or their vicinity in specific body regions in goats. Although chemical identity of the pheromone is yet to be determined, analyses of male goat hair extracts by gas chromatography fractionation suggest that the male effect pheromone is a volatile substance with relatively small molecular weight. From morphological and molecular biological studies in goats, it is suggested that the pheromone molecule is detected by a member of the V1R family located on both the olfactory neurones and the vomeronasal sensory neurones, and the pheromone signal is conveyed to the medial nucleus of amygdala via the main olfactory and vomeronasal pathways and, subsequently, to the hypothalamic GnRH pulse generator to enhance its activity.

Histamine ameliorates anti-glomerular basement membrane antibody-induced glomerulonephritis in rats.

Anti-glomerular basement membrane (anti-GBM)-induced glomerulonephritis involves T-helper type 1 (Th1) responses leading to rapid crescent formation. As many inflammatory and immune responses in general are affected by histamine, we examined the effects of histaminergic ligands on immune renal injury in the rat. Female Wistar-Kyoto rats were injected intraperitoneally with an antibody against the GBMs. Histaminergic ligands were then injected twice daily for 5 days after which renal function was assessed by proteinuria. Treatment with histamine led to significant dose-dependent reductions in proteinuria compared to the control antibody-injected group and markedly decreased the number of crescentic glomeruli and macrophage infiltration of the glomeruli. Furthermore, histamine significantly decreased the plasma concentration of interleukin-12, a Th1-type cytokine compared to the antibody-injected control animals. Dimaprit, an H(2)/H(4) agonist, mimicked the effects of histamine on proteinuria and crescent formation. Clozapine, an H(4) agonist, tended to mimic the effects of histamine, whereas an H(1), mepyramine, or an H(2) antagonist, ranitidine, did not reverse the protective effect of histamine. We suggest that histamine may alleviate renal injury in anti-GBM glomerulonephritis by suppressing the immune response.

Changes in the leukocyte distribution and surface expression of adhesion molecules induced by hypothalamic stimulation in the cat.

Emotions and the neuroendocrine system are known to affect leukocyte distribution. However, there have so far been few reports on the relationship between hypothalamically induced emotional behavior and the endocrine-immune response. We previously reported changes in the leukocyte distribution and adhesion molecules induced by anteromedial hypothalamus stimulation (AH stimulation), which elicits restlessness behaviors in the cat. In this study, we examined ventromedial hypothalamus stimulation (VMH stimulation), which elicits threat behaviors. In addition, the endocrine responses after VMH stimulation were evaluated. VMH stimulation as well as AH stimulation induced elevations of plasma cortisol and epinephrine levels and granulocytosis and lymphopenia. In contrast, VMH stimulation induced only an elevation of plasma norepinephrine and elicited an opposite pattern of CD62L expression on the leukocyte subpopulations. The different endocrine-immunological reactions between VMH stimulation and AH stimulation were thus associated with different types of behavioral responses.

Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation.

The loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that uses only one type of enzyme. One of the characteristics of the LAMP method is its ability to synthesize extremely large amount of DNA. Accordingly, a large amount of by-product, pyrophosphate ion, is produced, yielding white precipitate of magnesium pyrophosphate in the reaction mixture. Judging the presence or absence of this white precipitate allows easy distinction of whether nucleic acid was amplified by the LAMP method. Since an increase in the turbidity of the reaction mixture according to the production of precipitate correlates with the amount of DNA synthesized, real-time monitoring of the LAMP reaction was achieved by real-time measurement of turbidity.

Comparative effects of dietary fat types on hepatic enzyme activities related to the synthesis and oxidation of fatty acid and to lipogenesis in rats.

The effects of different types of dietary fat on the activities of hepatic enzymes related to fatty acid synthesis [glucose-6-phosphate dehydrogenase (G6PDH) and acetyl-CoA carboxylase (ACC)], oxidation [acyl-CoA synthetase (AST), carnitine palmitoyl transferase (CPT), and peroxisomal beta-oxidation (PbetaOX)], and lipogenesis [phosphatidate phosphohydrolase (PAP), diacylglycerol acyltransferase (DGAT), and phosphocholine diacylglycerol transferase (PCDGT)], and plasma and liver lipid levels were investigated in male Wistar rats. The animals were 6 weeks old and about 120 g of body weight, and were fed on test diets containing 20% of a mixture of tripalmitin, tristearin and corn oil (SFA), olive oil (OLI), sunflower oil (SUN), linseed oil (LIS), and sardine oil (SAR) for 2 weeks. The concentrations of plasma total cholesterol (T-CHOL), high-density lipoprotein-cholesterol (HDL-CHOL), triacylglycerol (TG) and phospholipid (PL) were generally higher in the rats fed on SFA and OLI than in those given SUN, LIS and SAR. The rats fed on OLI had a higher level of liver T-CHOL than those fed on the other fats. The liver TG content was nearly higher from the intake of SFA and OLI than from SUN, LIS and SAR, although the liver PL level was not affected by the type of dietary fat. The SFA and OLI groups had the highest activities of hepatic G6PDH and ACC, and the SAR group, the lowest activities. The activities of AST and CPT, and peroxisomal PbetaOX in the liver were higher in the rats fed on the LIS and SAR diets than in those given the other diets. The hepatic PAP activity was higher from the intake of OLI and SUN, and tended to be higher from SFA than from LIS and SAR. The activity of liver DGAT was higher from SFA and inclined to be higher from OLI, SUN, and LIS than from SAR, while the PCDGT activity in the liver was not effected by the type of dietary fat. The concentrations of plasma and liver TG were generally positively correlated with the activities of liver enzymes related to the synthesis of fatty acids and lipids, and negatively with those involved in fatty acid oxidation. Based on these results, it is suggested that the levels of plasma and liver TG were controlled by different types of dietary fat through changes in the hepatic enzyme activities related to fatty acid synthesis, lipogenesis, and fatty acid oxidation.

Hypothalamically induced emotional behavior and immunological changes in the cat.

Numerous animal studies on the correlation between stress and immunity have been performed but few such studies have been made concerning the relationship between various kinds of stress-related emotional behavior and immunological changes. Electrical stimulation of the hypothalamus in cats elicits various emotional behaviors such as restlessness, defensive attack, defensive retreat and quiet biting attack. We examined changes in the lymphocyte proliferative responses and plasma cortisol level which accompanied such emotional behavior. A significant increase in plasma cortisol was observed in the restlessness, defensive attack and defensive retreat groups, but not in the quiet biting attack or non-response (control) groups. A significant increase in the lymphocyte proliferative responses to phytohemagglutinin (PHA) was observed in the restlessness and defensive attack groups but not in the defensive retreat, quiet biting attack or non-response groups. These results suggest that various kinds of emotional behavior appear to be differentially correlated with the lymphocyte proliferative responses, while also being differentially correlated with the plasma cortisol concentration. Because the changes in lymphocyte responses and plasma cortisol did not always completely correlate with one another, the changes in the lymphocyte responses are not considered to be influenced by plasma cortisol alone.

Evidence in support of a four transmembrane-pore-transmembrane topology model for the Arabidopsis thaliana Na+/K+ translocating AtHKT1 protein, a member of the superfamily of K+ transporters.

The Arabidopsis thaliana AtHKT1 protein, a Na(+)/K(+) transporter, is capable of mediating inward Na(+) currents in Xenopus laevis oocytes and K(+) uptake in Escherichia coli. HKT1 proteins are members of a superfamily of K(+) transporters. These proteins have been proposed to contain eight transmembrane segments and four pore-forming regions arranged in a mode similar to that of a K(+) channel tetramer. However, computer analysis of the AtHKT1 sequence identified eleven potential transmembrane segments. We have investigated the membrane topology of AtHKT1 with three different techniques. First, a gene fusion alkaline phosphatase study in E. coli clearly defined the topology of the N-terminal and middle region of AtHKT1, but the model for membrane folding of the C-terminal region had to be refined. Second, with a reticulocyte-lysate supplemented with dog-pancreas microsomes, we demonstrated that N-glycosylation occurs at position 429 of AtHKT1. An engineered unglycosylated protein variant, N429Q, mediated Na(+) currents in X. laevis oocytes with the same characteristics as the wild-type protein, indicating that N-glycosylation is not essential for the functional expression and membrane targeting of AtHKT1. Five potential glycosylation sites were introduced into the N429Q. Their pattern of glycosylation supported the model based on the E. coli-alkaline phosphatase data. Third, immunocytochemical experiments with FLAG-tagged AtHKT1 in HEK293 cells revealed that the N and C termini of AtHKT1, and the regions containing residues 135-142 and 377-384, face the cytosol, whereas the region of residues 55-62 is exposed to the outside. Taken together, our results show that AtHKT1 contains eight transmembrane-spanning segments.

A possible role of neuropeptide Y as a mediator of undernutrition to the hypothalamic gonadotropin-releasing hormone pulse generator in goats.

To understand central mechanisms for nutritional infertility, the activity of the GnRH pulse generator was directly assessed in ovariectomized (OVX) goats under several experimental conditions by recording characteristic increases in the multiple-unit activity (volleys). When estradiol (E(2))-treated animals were fasted for 4-5 days, the activity of the GnRH pulse generator was gradually suppressed, and the volley interval at the end of fasting was significantly prolonged, compared with that during the feeding period (67.4 vs. 49.3 min, n = 5, P < 0.01). On the other hand, such a significant effect on the pulse generator was not observed in OVX goats. In the second experiment, the animals received a bolus intracerebroventricular injection of several doses (0, 2, 5, and 20 microg/400 microl) of neuropeptide Y (NPY). Exogenous NPY dose-dependently inhibited the pulse generator activity. At the highest dosage, the 1st posttreatment volley interval was significantly longer than that of the pretreatment (112.4 vs. 32.6 min, n = 5, P < 0.01) in OVX goats. The suppressive effect of NPY was similarly observed in OVX+E(2) goats. Further, when NPY was infused (10 microg/200 microl.h for 6 h) into OVX goats, the activity of the GnRH pulse generator was almost completely inhibited during the infusion period. Hypothalamic sites responding to fasting were immunohistochemically evaluated using an antibody for Fos in castrated goats. Fos-immunoreactive neurons were found in areas adjacent to the third ventricle. Double-labeling immunohistochemistry revealed that a subpopulation of NPY neurons in the arcuate nucleus was activated in response to fasting. These results demonstrate that: 1) the activity of the GnRH pulse generator is suppressed by fasting in the presence of E(2); 2) exogenous NPY inhibits the activity of the GnRH pulse generator regardless of the presence of E(2); and 3) several hypothalamic neurons or regions, including those containing NPY in the arcuate nucleus, are activated by fasting. Collectively, these observations suggest that NPY acts as a mediator of undernutrition to the GnRH pulse generator.

Radioimmunoassay for somatostatin receptor type 2.

OBJECTIVE: To develop radioimmunoassay for somatostatin receptor type 2 (SSTR2) and search for its presence in certain rat tissues. METHODS: Anti-SSTR2 antiserum has been raised in New Zealand white rabbits immunized with a conjugate of synthetic SSTR2 with bovine serum albumin. Radioiodination of SSTR2 was performed by chloramin T method followed by purification of radioiodinated material on Sephadex G-25 column. RESULTS: The obtained antibody did not crossreact with SSTR1, SSTR3, SSTR4, SSTR5, hypothalamic hormones, pituitary hormones, neuropeptides or gut hormones. The assay was performed with a double antibody system. SSTR2 was extracted from the tissues with acid acetone. The dilution curve of acid acetone-extracts of rat hypothalamus in the radioimmunoassay system was parallel to the standard curve. The recovery of tissue SSTR2 was about 89 %, and the intra-assay and inter-assay variations were 4.9 % and 7.8 %, respectively. SSTR2 was found in the hypothalamus, cerebrum, cerebellum, pituitary, stomach and testis. CONCLUSIONS: These data suggest that this assay system is suitable for the estimation of SSTR2 in the tissues.

How to distinguish garlic from the other Allium vegetables.

The establishment of international monographs for herbs is in progress. Here, we propose both a marker compound and a method for its analysis for the identification of garlic bulbs and their products. The constituents in 26 kinds of fresh edible parts of Allium vegetables and three types of garlic preparations were analyzed. Sulfur compounds are the most characteristic constituents in garlic, but manufacturing processes of garlic products dramatically affect these constituents. Thus, no sulfur compound could be specified as a universal marker of identification applicable for any type of garlic. On the other hand, garlic contains other characteristic compounds, namely, saponins. After analyzing Allium vegetables and garlic preparations, we concluded that sapogenins, especially beta-chlorogenin, may be a viable candidate for identifying and distinguishing garlic from other Allium vegetables.

Preparation of a monoclonal antibody to N(epsilon)-(Hexanonyl)lysine: application to the evaluation of protective effects of flavonoid supplementation against exercise-induced oxidative stress in rat skeletal muscle.

The monoclonal antibody to N(epsilon)-(hexanonyl)lysine (HEL), a novel adduct formed by the reaction of linoleic acid hydroperoxide and lysine, has been prepared and characterized. The obtained antibody specifically recognized the HEL moiety. Using the monoclonal antibody, we evaluated the protective effects of feeding eriocitrin, which is one of flavonoids in lemon fruit, on oxidative modification induced by exercise in rats. The supplementation of eriocitrin significantly suppressed the increase in HEL in the skeletal muscle by exercise. The result suggests that the determination of HEL may be a good method for evaluation of the protective effect of beneficial food factors against oxidative stress.

[Treatment of 522 patients with sudden deafness performed oxygenation at high pressure].

INTRODUCTION: Oxygenation at high pressure (OHP) is thought to be useful, even though regional blood flow is decreased, because increasing dissolved oxygen prevents the death of nerve tissue. In this report, we retrospectively investigated the effect of OHP on sudden deafness. OBJECT AND METHOD: We reviewed 522 patients treated with OHP at Kagawa Rosai Hospital over a ten-year period (January 1989 to December 1998). We discussed some prognostic factors: comparison between cases which had been treated with OHP previously and those which had not, number of days between onset and beginning of the treatment which included OHP, age, initial averaged five-frequency hearing level, vertigo, tinnitus, complications of OHP, cases of relapse and the time of the onset, which is about season, month and week. OHP was administered at a pressure of 2.5 atmospheres for 80 minutes a day from 10 to 15 times. All patients also received a course of intravenous administration of steroid, vitamin B12, Prostaglandin E1, ATP, and low-molecular dextran. RESULTS: Overall, complete recovery occurred in 19.7% of the patients, definite improvement in 34.9% (complete recovery included), and slight improvement in 58.1% (definite improvement included). Most of the patients (78.0%) were referred by other hospitals, because our hospital was the only one in the Sikoku area which had a big equipment of OHP. All 161 patients had already been treated in other hospitals over 8 days, but they had shown little improvement after the initial therapy. Of this group, complete recovery after the second course of treatment occurred in 13.0% of the patients, definite improvement in 19.3%, and slight improvement in 39.1%. OHP was thus effective for about 40% of patients who had been unresponsive to the initial therapy. Delay in treatment usually produces poor hearing recovery. There was a significant difference between those patients treated within 14 days and those treated 15 days or more after onset. The improvement rate also decreased with age. The prognosis of patients with vertigo was worse than those without vertigo. Tinnitus had no influence on the prognosis. There were no severe complications during the course of OHP, but otitis media with effusion occurred in 90 patients, and paracentesis was performed for 53 patients. CONCLUSION: The treatment of sudden deafness with OHP has been discussed in this report. Important prognostic factors were time between onset and beginning of the treatment which included OHP, age, vertigo, and the initial averaged five-frequency hearing level. We conclude that OHP should be performed within 14 days from onset, and that OHP was able to achieve hearing improvement in many cases unresponsive to the initial therapy if it was performed very early.

Radioimmunoassay for hypocretin-2.

OBJECTIVE: To develop radioimmunoassay for hypocretin-2 (Hcrt-2). And search for its presence in certain rat tissues. METHODS: Anti-Hcrt-2 serum has been raised in New Zealand white rabbits immunized with a conjugate of synthetic Hcrt-2 with bovine serum albumin. Radioiodination of Hcrt-2 was performed by chloramine T method, followed by purification of radoiodinated material on Sephadex G-25 column. RESULTS: The obtained antibody did not cross react with hypocretin-2, hypothalamic hormones, pituitary hormones, neuropeptides or gut hormones. The assay was performed with a double antibody system. Hcrt-2 was extracted from the tissues with acid acetone. The dilution curve of acid acetone extracts of rat hypothalamus in the radioimmunoassay system was parallel to the standard curve. The recovery of tissue Hcrt-2 was about 85 % and the intra-assay and inter-assay variation were 5.6 % and 8.0 %, respectively. Hcrt-2 was found in the hypothalamus, cerebrum, brain stem and testes. CONCLUSIONS: The obtained data suggest that the assay system developed is suitable to measure Hcrt-2 in tissues and that Hcrt-2 is mainly found in the hypothalamus.

Changes in the leukocyte distribution and surface expression of adhesion molecules accompanied with hypothalamically induced restlessness in the cat.

One type of emotional behavior called restlessness occurs when the anteromedial hypothalamus is stimulated in cats. We examined the changes in the distribution and surface expression of adhesion molecules in leukocytes accompanied with restlessness. Mature female cats were used for this study. The cats were stimulated with 60 Hz sine wave train pulses (20-90 microA, 10 s in duration, at 5-min intervals) for 60 min. Samples of blood were collected from 30 min before stimulation up to several hours after the final stimulation. The number of granulocytes increased just after stimulation, while at the same time the expression of L-selectin decreased. On the other hand, the number of CD4+ and CD8+ T lymphocytes decreased at 1-2 h after the end of the stimulation, while the expression of L-selectin increased. In addition, the expression of LFA-1 and VLA-4 did not change. These data suggest that hypothalamically elicited restlessness is thus accompanied by a leukocyte distribution change, which might be mediated by changes in the expression of L-selectin on leukocytes. Plasma cortisol increased during stimulation in restlessness. However, during in vitro culture experiments, cortisol did not alter the expression of leukocyte L-selectin which thus indicated that cortisol does not directly affect the surface expression of L-selectin. These results thus suggest that hypothalamically induced restlessness is a useful stress model for psychoneuroimmunological studies.

Effects of both the emotional behavior and feeding conditions on the circulating plasma volume and plasma glucose levels in cats.

Influence of hypothalamically induced emotional behavior on the circulating plasma volume, plasma levels of glucose, epinephrine (E), norepinephrine (NE), dopamine (DA) and cortisol were examined in awake cats under both fasted and fed conditions. Restlessness was evoked intermittently for 6 h by electrical stimulation of the anteromedial hypothalamus (AMH). Blood was sampled immediately before, 1 h after and 6 h after the start of stimulation. Changes in the plasma volume was calculated by changes of hemoglobin (Hb) and hematocrit (Ht). As the control group, another 7 cats with electrodes implanted but unstimulated were identically treated under both fasted and fed conditions. Both E and glucose levels in restlessness group once markedly increased after 1 h and then tended to decrease after 6 h, whereas NE levels in restlessness group increased after 1 h and further increased after 6 h, whether cats were fasted or fed. DA levels increased under the fasted condition of restlessness. The cortisol level markedly increased in both fasted and fed restlessness groups. The plasma volume in control group increased under the fed condition, while in restlessness group it decreased remarkably and tended to decrease more in a fasted state than in a fed state. These results indicated that AMH induced restlessness elicited marked sympatho-adrenal activation, hyperglycemia and hemoconcentration, whether cats were fasted or fed. Relationship among such responses, and the difference in responses between fasted and fed conditions were also discussed in the paper.

Effects of orexin A on thyrotropin-releasing hormone and thyrotropin secretion in rats.

Effects of orexin A on secretion of thyrotropin-releasing hormone (TRH) and thyrotropin (TSH) in rats were studied. Orexin A (50 microg/kg) was injected iv, and the rats were serially decapitated. The effects of orexin A on TRH release from the rat hypothalamus in vitro and on TSH release from the anterior pituitary in vitro were also investigated. TRH and thyroid hormone were measured by individual radioimmunoassays. TSH was determined by the enzyme-immunoassay method. The hypothalamic TRH contents increased significantly after orexin A injection, whereas its plasma concentrations tended to decrease, but not significantly. The plasma TSH levels decreased significantly in a dose-related manner with a nadir at 15 min after injection. The plasma thyroid hormone levels showed no changes. TRH release from the rat hypothalamus in vitro was inhibited significantly in a dose-related manner with the addition of orexin A. TSH release from the anterior pituitary in vitro was not affected with the addition of orexin A. The findings suggest that orexin A acts on the hypothalamus to inhibit TRH release.

Molecular and functional characterization of a novel mouse transient receptor potential protein homologue TRP7. Ca(2+)-permeable cation channel that is constitutively activated and enhanced by stimulation of G protein-coupled receptor.

Characterization of mammalian homologues of Drosophila transient receptor potential protein (TRP) is an important clue to understand molecular mechanisms underlying Ca(2+) influx activated in response to stimulation of G(q) protein-coupled receptors in vertebrate cells. Here we have isolated cDNA encoding a novel seventh mammalian TRP homologue, TRP7, from mouse brain. TRP7 showed abundant RNA expression in the heart, lung, and eye and moderate expression in the brain, spleen, and testis. TRP7 recombinantly expressed in human embryonic kidney cells exhibited distinctive functional features, compared with other TRP homologues. Basal influx activity accompanied by reduction in Ca(2+) release from internal stores was characteristic of TRP7-expressing cells but was by far less significant in cells expressing TRP3, which is structurally the closest to TRP7 in the TRP family. TRP7 induced Ca(2+) influx in response to ATP receptor stimulation at ATP concentrations lower than those necessary for activation of TRP3 and for Ca(2+) release from the intracellular store, which suggests that the TRP7 channel is activated independently of Ca(2+) release. In fact, TRP7 expression did not affect capacitative Ca(2+) entry induced by thapsigargin, whereas TRP7 greatly potentiated Mn(2+) influx induced by diacylglycerols without involvement of protein kinase C. Nystatin-perforated and conventional whole-cell patch clamp recordings from TRP7-expressing cells demonstrated the constitutively activated and ATP-enhanced inward cation currents, both of which were initially blocked and then subsequently facilitated by extracellular Ca(2+) at a physiological concentration. Impairment of TRP7 currents by internal perfusion of the Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid revealed an essential role of intracellular Ca(2+) in activation of TRP7, and their potent activation by the diacylglycerol analogue suggests that the TRP7 channel is a new member of diacylglycerol-activated cation channels. Relative permeabilities indicate that TRP7 is slightly selective to divalent cations. Thus, our findings reveal an interesting correspondence of TRP7 to the background and receptor stimulation-induced cation currents in various native systems.

Electrophysiological correlates of pulsatile and surge gonadotrophin secretion.

The hypothalamic gonadotrophin-releasing hormone (GnRH) pulse generator governs intermittent discharges of GnRH into the pituitary portal circulation and, consequently, modulates the pulsatile pattern of gonadotrophin secretion. Electrophysiological correlates of pulsatile gonadotrophin secretion have been demonstrated in the mediobasal hypothalamus of monkeys, rats and goats by recording multiple unit activity. A temporal coincidence between characteristic increases in multiple unit activity and gonadotrophin pulses in the circulation is seen under a variety of physiological and experimental conditions in all three species examined, providing evidence that hypothalamic multiple unit activity originates in the GnRH pulse generator. During a preovulatory gonadotrophin surge induced by oestrogen in ovariectomized animals or occurring spontaneously in intact animals, GnRH pulse generator activity is decelerated, suggesting that it is not involved in generating the gonadotrophin surge. The gonadotrophin surge may be generated by an oestrogen-responsive neuronal complex intrinsically different from the GnRH pulse generator, the electrical operation of which remains unknown.