Effect of human defensins on the cytoplasmic Ca2+ content in platelets

The effect of the total fraction of human defensins (HNP-1, HNP-2, and HNP-3) on the cytoplasmic Ca²⁺ content ([Ca²⁺]_i) in the platelets of healthy donors was studied. At concentrations of 0.1–40 μg/ml and an incubation time of 10 min defensins have no effect on [Ca²⁺]_i in platelets labeled with Fura-2AM. However, at higher concentrations (100 μg/ml) they increased platelet [Ca²⁺]_i. In addition, defensins (40 μg/ml) inhibited the Ca²⁺ increase in platelets induced by thrombin, adenosine diphosphate, and the lipopolysaccharide ofS. typhimurium endotoxin. The most pronounced inhibitory effect was observed in a suspension of thrombin-stimulated platelets. It is shown that the effect of human defensins on the functional activity of platelets is due to the alterations in the intracellular Ca²⁺. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 12, pp. 600–603, December, 1994     

Electrical stimulations of perifused magnocellular nuclei in vitro elicit Ca2+-dependent, tetrodotoxin-insensitive release of oxytocin and vasopressin

Isolated rat paraventricular (PVN) and supraoptic (SON) nuclei were perifused in vitro and oxytocin and vasopressin releases were measured by radioimmunoassay during rest and during electrical stimulation. Stimulations at a frequency of 10 Hz (10-s bursts, every 10 s for 5 min) and an intensity of 4 mA, induced significant hormone release only with long duration pulses (10 ms). Short pulses (1 ms) applied at various frequencies (10, 20, 40 or 80 Hz) and intensities (4, 5, 10 or 20 mA) had no effect. The electrically evoked release of both hormones was not affected by tetrodotoxin (TTX), a sodium channel blocker, but was blocked in low-calcium medium or in the presence of gallopamil hydrochloride (D-600), a calcium channel blocker. These results suggest that, following electrical stimulation, oxytocin and vasopressin are released locally within the magnocellular nuclei even when blocking action potentials. The possibility of dendritic release is discussed.     

The effect of arginine vasopressin on the autologous mixed lymphocyte reaction.

Arginine vasopressin (AVP) is a nonapeptide that has been shown to be released from the supraoptic and paraventricular nuclei of the hypothalamus during stress. Although noted primarily for its hemodynamic as well as homeostatic properties, AVP also appears to have an effect on the immune system. It may modulate cellular immunity via its enhancement of the autologous mixed lymphocyte response (AMLR), an effect which we have demonstrated to occur over a wide dose range with a maximum at 10(-7) M. The increase in proliferation following a single addition of AVP in a 6-day culture appears to be augmented when the peptide is added daily throughout the same culture period. Enhanced proliferation appears to be a specific response that is influenced by arginine residues in position 8 of this nonapeptide. Having provided evidence for the existence of receptors with moderate affinity for AVP, we suggest a potential modulatory role for AVP in support of the concept of a communication between the neuroendocrine and immune systems. Since various autoimmune conditions may be aggravated by stress, stress-induced release of neuropeptides such as AVP may play an important role in modulating immune regulation of these disease states.     

Mobilization of intracellular Ca2+ induced by ADP and thrombin in platelets from diabetic patients with vascular complications

It is shown that the baseline level of cytoplasmic Ca²⁺ in platelets from diabetic patients is nearly 1.5 times as high as in healthy donors. The thrombin-induced increase of intracellular Ca²⁺ in patients with angiopathies is reliably lower than in the control, while in patients without angiopathies it is higher than that in donor platelets. The evevation of cytoplasmic Ca²⁺ induced by ADP is greater in both groups of patients. No Changes are found in the baseline level of intracellular Ca²⁺ or in the ADP-induced concentration of Ca²⁺ in platelets from diabetic patients during a 12-week course of insulin therapy. The intracellular Ca²⁺ does not rise after 2 weeks of insulin treatment in platelets from diabetic patients. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 121, N ^o 1, pp. 112–115, January, 1996     

Urinary excretion of angiotensin I, II, arginine vasopressin and oxytocin in patients with anaphylactoid reactions

Human urine samples, purified on octadecasilyl-silica cartridges, contained immunoreactive angiotensin I, II, arginine vasopressin and oxytocin. The daily excretion of these peptides in healthy volunteers was 190.00 +/- 38.43 (n = 12), 17.48 +/- 3.09 (n = 12), 63.43 +/- 14.84 (n = 8) and 13.52 +/- 1.42 (n = 7) pmol/24 hr, respectively (mean +/- s.e.m.). Patients with a history of anaphylactoid reactions to drugs or food additives showed clinical symptoms such as urticaria, flush, nausea, dizziness and hypotension after oral provocation with cyanocobalamine, propyphenazone, acetylsalicylic acid and sodium benzoate. In five of the seven patients, angiotensin I and II were increased several fold in the urine fractions after symptoms were reported. The average increase in the urine concentration of both peptides was fourfold and 5.5-fold. In three out of five patients, the mean excretion of arginine vasopressin and oxytocin immunoreactive material was also elevated by a factor of 5.7 and 4.4, respectively. Oral provocation with a placebo failed to elicit anaphylactoid symptoms or an increase in the urine levels of angiotensin I or angiotensin II. Angiotensin I and angiotensin II-like immunoreactivity could be characterized on HPLC as Ile5-angiotensin I, Ile5-angiotensin II and angiotensin II metabolites. HPLC characterization of immunoreactive arginine vasopressin and oxytocin in two different gradient systems showed retention times different than the retention times of the corresponding synthetic standard peptides indicating that both peptides are not authentic AVP and OXT. These results suggest that angiotensin I and angiotensin II may be involved in the clinical events observed during some forms of anaphylactoid reactions.     

精氨酸加压素在内毒素热限形成中的作用

本文观察了家兔静脉注射不同剂量ＥＴ后中隔区，下丘脑组织及血浆中ＡＶＰ含量的变化。结果显示：在ＥＴ发热过程中，中隔区，下丘脑及血浆ＡＶＰ含量均显著增多（Ｐ＜０．０１）；ＥＴ发热达热限时，体温不再升高，中隔区与血浆ＡＶＰ含量也不再增多，且中隔区及血浆ＡＶＰ含量变化与体温变化呈明显正相关（ｒ＝０．９８４，０．０５＜Ｐ＜０．０１；ｒ＝０．９９４，Ｐ＜０．０１）；此时，下丘脑升高的ＡＶＰ含量开始下降（Ｐ０。     

ADP exerts a protective effect against rundown of the Ca2+ current in bovine chromaffin cells

In isolated chromaffin cells, the high-voltage-activated Ca²⁺ current, recorded using 5 mM Ca²⁺ as the divalent charge carrier, exhibits rundown within 10 min, which is delayed for 1 h at least by the addition of 1 mM adenosine 5-triphosphate (ATP) to the pipette medium. The mechanism of this stabilizing action of ATP has been examined. ATP action is dose dependent; the rundown process, which was delayed at concentrations below 0.4 mM, was totally abolished at higher concentrations. The requirement for ATP was shown to be quite strict: 2 mM inosine 5-triphosphate (ITP) could not replace ATP, whereas guanosine 5-triphosphate (GTP) could, but at higher concentrations. This effect of ATP was shown to require the presence of MgCl₂ and the liberation of a phosphate group since the ATP analogue 5-adenylyl-imidodiphosphate (AMP-PNP) could not act as a substitute for ATP, suggesting an action through either adenosine 5-diphosphate (ADP) or a phosphorylation step. ADP, in the presence of Mg²⁺ only, could replace ATP in the same concentration range. This effect was shown to be specific to ADP; it was maintained after blocking the pathways which convert ADP into ATP, and could not be mimicked by guanosine 5-diphosphate (GDP). Similarly, ATP and ADP effects were abolished at an increased internal Ca²⁺ concentration (pCa 6 instead of pCa 7.7, where pCa = –log₁₀[Ca²⁺]). Nevertheless, the presence of 1 mM Mg-ADP in the bathing solution did not prevent the rundown of the Ca²⁺ channels when going to the inside-out patch recording configuration. In conclusion, the stabilizing effect of ATP may be interpreted by a Mg²⁺-ADP binding site present on high-voltage-activated Ca²⁺ channels. A localization of such an ADP regulatory site on the L-type Ca²⁺ channel itself cannot be excluded, though with an additional requirement since Mg-ADP alone is not able to maintain the corresponding activity on excised patches.     

Association of fibrinogen with human platelets pretreated with chymotrypsin or aggregated with ADP or thrombin: an immunocytochemical study.

Although platelets can be induced to aggregate in the absence of external fibrinogen, the response is greatly potentiated by fibrinogen and fibrinogen becomes associated with the surface of stimulated platelets. We compared the aggregation response and association of fibrinogen with the surface of platelets aggregated by ADP or thrombin, and of chymotrypsin-treated platelets aggregated by fibrinogen. The association of fibrinogen with the surface of the platelets was visualized using an electron microscope immunocytochemical method. The aggregation response and the pattern of fibrinogen association was different with each of the three agonists studied. ADP-induced aggregation was associated with pseudopod formation and fibrinogen binding; granule contents were not released and aggregation and fibrinogen binding were reversible. Thrombin-induced aggregation was associated with extensive pseudopod formation and the release of granule contents, but platelet-to-platelet adherence did not appear to involve fibrinogen binding at sites remote from regions of granule discharge; disaggregation did not occur, and visible fibrin did not form rapidly in the absence of added fibrinogen. Fibrinogen-induced aggregation/agglutination of chymotrypsin-treated platelets was similar to ADP-induced aggregation in that fibrinogen binding was required and granule contents were not released; it differed from ADP-induced aggregation in that pseudopod formation did not occur and the aggregates were irreversible. Fibrinogen-induced aggregation of chymotrypsin-treated platelets differed from thrombin-induced aggregation of untreated platelets in every respect except irreversibility. Thus neither pseudopod formation, fibrinogen binding nor the release of granule contents is essential for platelet-to-platelet adherence, although one or other or all may occur in association with it. If platelets are not stimulated to release their granule contents, fibrinogen binding appears to be necessary for extensive platelet aggregation.     

Effects of vasopressin and urea on Ca2+-calmodulin-dependent renal prostaglandin E

Vasopressin (AVP stimulated immunoreactive E (iPGE) synthesis and the release of [3H]arachidonate (AA) from prelabeled slices of rat inner medulla (IM) in the presence but not in the absence of Ca2+ (plus 2 mM EGTA). Urea (700-1,200 mosM) inhibited these actions. By contrast Ca2+ deprivation or urea did not suppress AVP-induced increases in cAMP or stimulation of iPGE by exogenous AA. At 10-50 microM, trifluoperazine (TFP) suppressed AVP-induced increases in [3H]AA release and iPGE accumulation, but not increases in cAMP. Basal acyl hydrolase activity (AH) of 2,000 g particulate fractions of IM was suppressed by EGTA. Ca2+, but not Mg2+ or AVP, restored AH to levels observed in the absence of EGTA. Ca2+-induced increases in particulate AH were inhibited by urea (700-1,200 mosM) or TFP (10-50 microM), but not 1,000 mosM NaCl. Partial depletion of particulate calmodulin-like activity suppressed Ca2+-induced increases in AH. The latter was restored with 1 microM purified exogenous calmodulin but not troponin C. The results demonstrate that AVP stimulation of AA and PGE release in IM are Ca2+-dependent processes suppressed by urea. They also suggest a role for Ca2+-calmodulin-dependent AH in the control of PG synthesis in IM.     

The effect of vasopressin and oxytocin on behavioral processes and brain neurotransmitter metabolism in rats

Neuroscience and Behavioral Physiology -     

The effect of arginine vasopressin and V1 receptor antagonist on brain water in cat

Arginine vasopressin (AVP) is important in brain water regulation. To better understand the effect of AVP released by extrahypothalamic fibers in brain, we microinfused AVP into intact brain and studied its effect on brain water and electrolytes. Adult cats had 5 ng of AVP infused into the caudate nuclei. Four h after infusion the brains were removed for measurement of water and electrolyte contents. Animals infused with AVP were compared to controls infused with saline. AVP increased water content significantly in gray and white matter sites, while electrolyte content was unchanged. Another group of animals had intracerebral infusions with 5 ng of AVP and 50 ng of a V1 receptor antagonist, (d(CH2)5Tyr-(Me)AVP). The antagonist blocked the increase in water, suggesting a V1 receptor mediated the action.     

Early stages in the mechanism of action of glucocorticoids on human platelets. Effect of hydrocortisone on intracellular cAMP and Ca2+ content

Changes in basal and stimulated levels of cAMP and calcium induced by hydrocortisone in a wide range of concentration (0.1–25 μM) are studies in a suspension of washed human platelets. The effects of hydrocortisone on the activity of preparations modulating various stages of the adenylate cyclase system (forskolin, adenosine, adrenaline, and 3-isobutyl-1-methylxanthine) are compared. Platelets are stimulated with collagen, platelet activating factor, and thapsigargin. Hydrocortisone in different concentrations acts as both activator and inhibitor of calcium metabolism in platelets. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 123, No. 6, pp. 663–665, June, 1997     

The effect of extracellular Ca2+ concentration on the negative staircase of Ca2+ transient in field-stimulated rat ventricular cells

We performed experiments using the Ca²⁺ indicator dye, fura-2 to investigate the effect of extracellular Ca²⁺ concentration ([Ca²⁺]_o) on sarcoplasmic reticulum (SR) Ca²⁺ release and loading in single rat ventricular cells. In normal Tyrode solution (1.8 mM [Ca²⁺]_o) repetitive stimulation (0.5 Hz) resulted in a gradual decrease in calcium transients (the negative staircase phenomenon) without being accompanied by a gradual decrease in diastolic intracellular Ca²⁺ concentration. The rate of the slow decline in calcium transient was faster in lower [Ca²⁺]_o. However, the peak of the first calcium transient was relatively invariant over a wide range of [Ca²⁺]_o (0.5–5 mM). The size of the calcium transient elicited by field stimulation was proportional to that induced by 10 mM caffeine, applied following the field stimulation. These results suggest that the size of calcium transients depends mainly on the Ca²⁺ content of the SR. The quiescent period favoured the replenishment of the SR and this effect was promoted further by increasing the driving force for Ca²⁺ entry across the sarcolemma during this period. We conclude that in low [Ca²⁺]_o, short stimulation interval may limit Ca²⁺ influx across the sarcolemma during the quiescent period to cause a gradual reduction in calcium content of the SR and thus the calcium transient.     

Glutamate receptor agonists increase intracellular Ca2+ independently of voltage-gated Ca2+ channels in rat cerebellar granule cells

Changes in membrane potential and cytosolic free Ca2+ concentrations, [Ca2+]i, in response to L-glutamate and glutamate receptor agonists were measured in rat cerebellar granule cells grown on coverslips. The membrane was depolarized by the application of L-glutamate and kainate, and by elevating the extracellular K+ concentration, as determined by using the membrane potential probe bisoxonol (DiBA-C4-(3)). The [Ca2+]i as measured with fura-2 was 220 nM on average under resting conditions and increased by raising the extracellular K+ and by applying L-glutamate, kainate, quisqualate or N-methyl-D-aspartate (NMDA). Verapamil and nifedipine reduced the high-K+ induced rise in [Ca2+]i but did not significantly affect the responses produced by NMDA, quisqualate and kainate, suggesting that the increase in intracellular Ca2+ in response to glutamate receptor agonists is primarily due to Ca2+ influx through receptor-coupled ion channels.     

The effect of cold exposure on fluid balance,circulating arginine vasopressin concentration and milk secretion in the goat

Total body water, water intake, urine output, milk yield, plasma, milk and urine osmolality and plasma arginine vasopressin concentration were measured in goast exposed to thermoneutral (20°C) and cold (0–1.0°C) environments for 24h. Cold exposure caused the animals to reduce their water intake substantially. This was accompanied by a decrease in total body water and an increase in osmolality of plasma and milk. The output of urine decreased as cold exposure progressed but free water clearance by the kidney was not significantly different in thermoneutral and cold environments and cold exposure had no effect on circulating arginine vasopressin concentration. Milk yield was reduced by cold exposure and it is suggested that the reduced net movement of water from blood to milk is partly a consequence of the dehydration in duced by cold exposure and that this, in turn, is due primarily to a decrease in water intake with no effective renal compensation.     

A quantitative analysis of the interrelationships between subpopulations of rat sensory neurons containing arginine vasopressin or oxytocin and those containing substance P, fluoride-resistant acid phosphatase or neurofilament protein

In rat L5 dorsal root ganglia 50% of neurons contained arginine vasopressin-like immunoreactivity and 38% oxytocin-like immunoreactivity, the oxytocin entirely coexisting with the arginine vasopressin. Staining of alternate mirror-image sections with RT97 (an antibody to neurofilament protein, and a marker for large light neurons) and with arginine vasopressin antiserum showed that the two were entirely complementary, thus establishing arginine vasopressin as a marker for all small dark neurons. Mirror-image staining also showed that neurons containing substance P-like immunoreactivity and those containing fluoride-resistant acid phosphatase activity were each contained within the arginine vasopressin-positive population. Arginine vasopressin-like immunoreactivity was axonally transported in the dorsal root and (in greater quantity) in sciatic nerve. Arginine vasopressin-like immunoreactivity was present also in laminae I and II of the dorsal horn of the spinal cord and this reactivity was absent in animals which had been treated neonatally with capsaicin, suggesting that it was contained in primary afferent terminals. These results are discussed in terms of their implications for the classification of primary afferent neurons and of a possible physiological role for arginine vasopressin in these neurons.     

Preimplantation embrology: The role of ryanodine-sensitive Ca2+ stores in the Ca2+ oscillation machine of human oocytes

This study was undertaken to localize ryanodine-sensitive Ca²⁺stores in human oocytes and to evaluate their role in the Ca²⁺oscillations responsible for oocyte activation at fertilization.The addition of ryanodine provoked a Ca²⁺ discharge from storeslocalized throughout the ooplasm with the exception of the corticaland subcortical peripheral regions. The ryanodine-induced dischargewas typically followed by a short series of Ca²⁺ oscillationsthat only involved the cytoplasmic region populated by the ryanodine-sensitivestores. In contrast, the Ca²⁺ oscillations induced by the thiolreagent thimerosal or by spermatozoa at fertilization were ofa much longer duration and also involved ryanodine-insensitivestores. Presumably, these ryanodine-insensitive stores are sensitiveto inositol 1, 4, 5-trisphosphate (InsP3). The addition of ryanodineto oocytes during ongoing thimerosal- or sperm-induced Ca²⁺oscillations inhibited the oscillations. These data suggesta co-operation between the ryanodine-sensitive and ryanodine-insensitivestores in maintaining the sperm-induced Ca²⁺ oscillations. Inthis two-store oscillation model, each periodic [Ca²⁺]i increaseis triggered by a Ca²⁺ discharge from the peripheral, InsP₃-sensitivestores inducing Ca²⁺-induced Ca²⁺ release from the ryanodine-sensitivestores. However, the pacemaker frequency of the Ca²⁺ dischargesfrom the InsP₃-sensitive stores is conditioned by the actualphysiological state of the ryanodine-sensitive stores. Ca²⁺ oscillations/Ca²⁺ stores/fertilization/ryanodine/thimerosal     

The effect of intracellular pH on cytosolic Ca2+ in HT29 cells

 The influence of intracellular pH (pH_i) on intracellular Ca²⁺ activity ([Ca²⁺]_i) in HT₂₉ cells was examined microspectrofluorometrically. pH_i was changed by replacing phosphate buffer by the diffusible buffers CO₂/HCO₃ ^–or NH₃/NH₄ ⁺ (pH 7.4). CO₂/HCO₃ ^–buffers at 2,5 or 10% acidified pH_i by 0.1, 0.32 and 0.38 pH units, respectively, and increased [Ca²⁺]_i by 8–15 nmol/l. This effect was independent of the extracellular Ca²⁺ activity and the filling state of thapsigargin-sensitive Ca²⁺ stores. Removing the CO₂/HCO₃ ^–buffer alkalinized pH_i by 0.14 (2%), 0.27 (5%), and 0.38 (10%) units and enhanced [Ca²⁺]_i to a peak value of 20, 65, and 143 nmol/l, respectively. Experiments carried out with Ca²⁺-free solution and with thapsigargin showed that the [Ca²⁺]_i transient was due to release from intracellular pools and stimulated Ca²⁺ entry. NH₃/NH₄ ⁺ (20 mmol/l) induced a transient intracellular alkalinization by 0.6 pHunits and increased [Ca²⁺]_i to a peak (Δ [Ca²⁺]_i = 164 nmol/l). The peak [Ca²⁺]_i increase was not influenced by removal of external Ca²⁺, but the decline to basal [Ca²⁺]_i was faster. Neither the phospholipase C inhibitor U73122 nor the inositol 1,4,5-trisphosphate (InsP ₃) antagonist theophylline had any influence on the NH₃/NH₄ ⁺-stimulated [Ca²⁺]_i increase, whereas carbachol-induced [Ca²⁺]_i transients were reduced by more than 80% and 30%, respectively. InsP ₃ measurements showed no change of InsP ₃ during exposure to NH₃/NH₄ ⁺, whereas carbachol enhanced the InsP ₃ concentration, and this effect was abolished by U73122. The pH_i influence on ”capacitative” Ca²⁺ influx was also examined. An acid pH_i attenuated, and an alkaline pH_i enhanced, carbachol- and thapsigargin-induced [Ca²⁺]_i influx. We conclude that: (1) an alkaline pH_i releases Ca²⁺ from InsP ₃-dependent intracellular stores; (2) the store release is InsP ₃ independent and occurs via an as yet unknown mechanism; (3) the store release stimulates capacitative Ca²⁺ influx; (4) the capacitative Ca²⁺ influx activated by InsP ₃ agonists is decreased by acidic and enhanced by alkaline pH_i. The effects of pH_i on [Ca²⁺]_i should be of relevance under many physiological conditions. Received: 17 June 1996 / Received after revision and accepted: 30 August 1996     

Apolipoprotein E genotype has no effect on the free intracellular Ca2+ concentration of platelets from Alzheimer's disease patients

The free intracellular Ca2+ concentration [Ca2+]i of platelets was investigated in Alzheimer's disease (AD) patients and age-matched control subjects with distinct Apo E genotypes. No significant differences were found between the Apo E genotype and the [Ca2+]i levels of platelets (basal and alpha-thrombin stimulated) from AD patients and age-matched control subjects, suggesting that [Ca2+]i homeostasis of platelets from AD patients is independent of the Apo E genotype. The results are discussed in terms of involvement of Apo E and [Ca2+]i changes in the etiopathogenesis of AD.