Platelet sarco/endoplasmic reticulum Ca2+ATPase isoform 3b and Rap 1b: interrelation and regulation in physiopathology

Platelet Ca2+ signalling involves intracellular Ca2+ pools, whose content is controlled by sarco/endoplasmic reticulum Ca2+ATPases (SERCAs). Among these, a key role is played by the inositol trisphosphate-sensitive Ca2+ pool, associated with the SERCA 3b isoform. We have investigated the control of this Ca2+ pool through the cAMP-dependent phosphorylation of the GTP-binding protein, Rap (Ras-proximate) 1b. We first looked for this Ca2+ pool target of regulation by studying the expression of the different SERCA and Rap 1 proteins in human platelets and various cell lines, by Western blotting and reverse transcription-PCR. Since co-expression of Rap 1b and SERCA 3b was obtained, we looked for their protein–protein interaction as a function of the cAMP-dependent phosphorylation of Rap 1b. Co-immunoprecipitations of SERCA 3b and Rap 1b proteins were found in the absence of phosphorylation, induced by the catalytic subunit of the cAMP-dependent protein kinase (csPKA). In contrast, upon pre-treatment of platelet membranes with csPKA, the SERCA 3b dissociated from the Rap 1b protein, in agreement with a role of its phosphorylated state in their interaction. Finally, we looked for adaptation of this complex in a platelet pathological model of hypertension. We investigated the expression of both proteins, as well as the cAMP-dependent phosphorylation of Rap 1b and SERCA 3b activity in platelets from control normotensive Wistar-Kyoto rats and from spontaneously hypertensive rats (SHRs). A decrease in SERCA 3b activity was associated with a decrease in Rap 1b endogenous phosphorylation in SHR platelets, consistent with a functional role in the regulation of the SERCA 3b-associated Ca2+ pool.

Download Full-text

MO094SALT INFLUENCES UROMODULIN EXCRETION INDEPENDENT OF BLOOD PRESSURE

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfab106.003 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

Sheon Mary ◽

Philipp Boder ◽

Giacomo Rossitto ◽

Lesley Graham ◽

Kayley Scott ◽

...

Keyword(s):

Blood Pressure ◽

Endoplasmic Reticulum ◽

Direct Effect ◽

Ex Vivo ◽

Mrna Levels ◽

Salt Loading ◽

Spontaneously Hypertensive ◽

Risk Variants ◽

Wistar Kyoto

Abstract Background and Aims Uromodulin (UMOD) is the most abundant renal protein secreted into urine by the thick ascending epithelial (TAL) cells of the loop of Henle. Genetic studies have demonstrated an association between UMOD risk variants and hypertension. Studies on UMOD overexpressing transgenic mice have shown that UMOD increases the tubular salt reabsorption via enhanced NKCC2 activity. We aimed to dissect the effect of salt-loading and blood pressure on the excretion of UMOD. Method Wistar-Kyoto (WKY) and stroke-prone spontaneously hypertensive (SHRSP) rats (n=8/sex/strain) were maintained on 1% NaCl for three weeks. Salt-loaded SHRSP were treated with nifedipine. Tubule isolation and ex vivo incubation with nifedipine were used to assess its direct effect on TAL. Results Urinary UMOD excretion was significantly reduced after salt loading in both strains (figure). In salt-loaded SHRSP, nifedipine treatment reduced blood pressure and urinary UMOD excretion. The reductions in urinary UMOD excretion were dissociated from unchanged kidney UMOD protein and mRNA levels, however, were associated with UMOD endoplasmic reticulum accumulation, thus suggesting secretion as a key regulatory step. Ex vivo experiments with TAL tubules showed that nifedipine did not have a direct effect on UMOD secretion. Conclusion Our data suggest a direct effect of salt on UMOD secretion independent of blood pressure and a potential role of endoplasmic reticulum stress on the control of UMOD secretion. The role of UMOD as a cardiovascular risk marker deserves mechanistic reappraisal and further investigations based on our findings.

Download Full-text

Increased Pressor Responsiveness to Enkephalin in Spontaneously Hypertensive Rats: The Role of Vasopressin

Clinical Science ◽

10.1042/cs059235s ◽

1980 ◽

Vol 59 (s6) ◽

pp. 235s-237s ◽

Cited By ~ 17

Author(s):

R. W. Rockhold ◽

J. T. Crofton ◽

L. Share

Keyword(s):

Blood Pressure ◽

Heart Rate ◽

Pressor Response ◽

Cardiovascular Effects ◽

Hypertensive Rats ◽

Methionine Enkephalin ◽

Spontaneously Hypertensive ◽

Wistar Kyoto Rats ◽

Wistar Kyoto

1. The cardiovascular effects of an enkephalin analogue were examined in spontaneously hypertensive and normotensive Wistar-Kyoto rats. (D-Ala2)-methionine enkephalin caused a biphasic increase in blood pressure and an increase in heart rate after intracerebroventricular injection. 2. The initial pressor response to (D-Ala2)-methionine enkephalin was greater in hypertensive than in normotensive rats. No difference was noted between groups during the secondary pressor response. Heart rate increases paralleled the secondary increase in blood pressure. 3. Naloxone pretreatment abolished the secondary increase in blood pressure and the tachycardia, but did not blunt the initial pressor response in female Wistar-Kyoto rats. 4. Plasma levels of arginine vasopressin were depressed during the plateau phase of the pressor response in hypertensive rats given intracerebroventricular (d-Ala2)-methionine enkephalin. 5. The results suggest that the cardiovascular effects of central enkephalin are not due to vasopressin, but may involve activation of the sympathetic nervous system.

Download Full-text

Role of adenosine in noradrenergic neurotransmission in spontaneously hypertensive rats

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1987.253.4.h909 ◽

1987 ◽

Vol 253 (4) ◽

pp. H909-H918 ◽

Cited By ~ 5

Author(s):

E. K. Jackson

Keyword(s):

Plasma Levels ◽

Spontaneously Hypertensive Rat ◽

Base Line ◽

Inhibitory Effects ◽

Spontaneously Hypertensive ◽

Vascular Responses ◽

Rat Mesentery ◽

Wistar Kyoto

The purpose of this study was to compare the in vivo role of adenosine as a modulator of noradrenergic neurotransmission in the spontaneously hypertensive rat (SHR) and Wistar-Kyoto control rat (WKY). In the in situ blood-perfused rat mesentery, vascular responses to periarterial (sympathetic) nerve stimulation (PNS) and to exogenous norepinephrine (NE) were enhanced in SHR compared with WKY. In both SHR and WKY, vascular responses to PNS were more sensitive to inhibition by adenosine than were responses to NE. At matched base-line vascular responses, compared with WKY, SHR were less sensitive to the inhibitory effects of adenosine on vascular responses to PNS, but SHR and WKY were equally sensitive with respect to adenosine-induced inhibition of responses to NE. Antagonism of adenosine receptors with 1,3-dipropyl-8-p-sulfophenylxanthine shifted the dose-response curve to exogenous adenosine sixfold to the right yet did not influence vascular responses to PNS or NE in either SHR or WKY. Furthermore, PNS did not alter either arterial or mesenteric venous plasma levels of adenosine in SHR or WKY, and plasma levels of adenosine in both strains were always lower than the calculated threshold level required to attenuate neurotransmission. It is concluded that in vivo 1) exogenous adenosine interferes with noradrenergic neurotransmission in both SHR and WKY; 2) SHR are less sensitive to the inhibitory effects of exogenous adenosine on noradrenergic neurotransmission than are WKY; 3) endogenous adenosine does not play a role in modulating neurotransmission in either strain under the conditions of this study; and 4) enhanced noradrenergic neurotransmission in the SHR is not due to defective modulation of neurotransmission by adenosine.

Download Full-text

Hypertension in SHR rats: contribution of maternal environment

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1987.253.4.h980 ◽

1987 ◽

Vol 253 (4) ◽

pp. H980-H984 ◽

Cited By ~ 6

Author(s):

M. A. Cierpial ◽

R. McCarty

Keyword(s):

Shr Rats ◽

Maternal Environment ◽

Spontaneously Hypertensive ◽

Arterial Blood ◽

Rearing Conditions ◽

Blood Pressures ◽

Wistar Kyoto ◽

Male Subjects ◽

Cross Fostering

The role of the maternal environment in the development of hypertension in spontaneously hypertensive (SHR) rats was evaluated using the technique of reciprocal cross fostering. Litters of SHR and Wistar-Kyoto (WKY) normotensive pups were either reared by their natural mothers, in fostered to mothers of the same strain, or cross fostered to mothers of the opposite strain shortly after birth. Litters were weaned at 21 days of age, at which time all pups were weighed. At 18-20 wk of age, resting mean arterial blood pressures (MAP) and heart rates were determined for male subjects from the six groups (2 strains X 3 rearing conditions) via an indwelling tail artery catheter. At weaning, SHR animals weighed less than WKY animals. SHRs fostered to WKY mothers were significantly heavier than control SHRs, and WKYs fostered to SHR mothers were significantly lighter than WKY controls at weaning. These body weight differences were also evident in adulthood. Cross fostering SHR pups to normotensive WKY mothers resulted in a dramatic reduction in resting MAP measured in adulthood. Conversely, cross fostering WKY pups to SHR mothers had no measurable effect on adult resting MAP. We propose that an interaction between characteristics of the SHR maternal environment and a genetic susceptibility in SHR pups is essential in triggering the full expression of the hypertensive phenotype in this animal model of human essential hypertension.

Download Full-text

PDK1 selectively phosphorylates Thr(308) on Akt and contributes to human platelet functional responses

Thrombosis and Haemostasis ◽

10.1160/th13-06-0484 ◽

2014 ◽

Vol 111 (03) ◽

pp. 508-517 ◽

Cited By ~ 30

Author(s):

Carol Dangelmaier ◽

Bhanu Kanth Manne ◽

Elizabetta Liverani ◽

Jianguo Jin ◽

Paul Bray ◽

...

Keyword(s):

Protein Kinase ◽

Protein A ◽

Human Platelets ◽

Clot Retraction ◽

Functional Responses ◽

Atp Secretion ◽

Dependent Protein ◽

Induced Aggregation

Summary3-phosphoinositide-dependent protein kinase 1 (PDK1), a member of the protein A,G and C (AGC) family of proteins, is a Ser/Thr protein kinase that can phosphorylate and activate other protein kinases from the AGC family, including Akt at Thr308, all of which play important roles in mediating cellular responses. The functional role of PDK1 or the importance of phosphorylation of Akt on Thr308 for its activity has not been investigated in human platelets. In this study, we tested two pharmacological inhibitors of PDK1, BX795 and BX912, to assess the role of Thr308 phosphorylation on Akt. PAR4-induced phosphorylation of Akt on Thr308 was inhibited by BX795 without affecting phosphorylation of Akt on Ser473. The lack of Thr308 phosphorylation on Akt also led to the inhibition of PAR4-induced phosphorylation of two downstream substrates of Akt, viz. GSK3β and PRAS40. In vitro kinase activity of Akt was completely abolished if Thr308 on Akt was not phosphorylated. BX795 caused inhibition of 2-MeSADP-induced or collagen-induced aggregation, ATP secretion and thromboxane generation. Primary aggregation induced by 2-MeSADP was also inhibited in the presence of BX795. PDK1 inhibition also resulted in reduced clot retraction indicating its role in outside-in signalling. These results demonstrate that PDK1 selectively phosphorylates Thr308 on Akt thereby regulating its activity and plays a positive regulatory role in platelet physiological responses.

Download Full-text

Suppression of endoplasmic reticulum stress improves endothelium-dependent contractile responses in aorta of the spontaneously hypertensive rat

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00952.2012 ◽

2013 ◽

Vol 305 (3) ◽

pp. H344-H353 ◽

Cited By ~ 59

Author(s):

Kathryn M. Spitler ◽

Takayuki Matsumoto ◽

R. Clinton Webb

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Spontaneously Hypertensive Rat ◽

Peripheral Resistance ◽

Spontaneously Hypertensive ◽

Cytosolic Phospholipase ◽

Hypertensive Rat ◽

Tert Butyl Hydroperoxide ◽

Extracellular Signal ◽

Wistar Kyoto

A contributing factor to increased peripheral resistance seen during hypertension is an increased production of endothelium-derived contractile factors (EDCFs). The main EDCFs are vasoconstrictor prostanoids, metabolites of arachidonic acid (AA) produced by Ca2+-dependent cytosolic phospholipase A2 (cPLA2) following phosphorylation (at Ser505) mediated by extracellular signal-regulated kinase (ERK1/2) and cyclooxygenase (COX) activations. Although endoplasmic reticulum (ER) stress has been shown to contribute to pathophysiological alterations in cardiovascular diseases, the relationship between ER stress and EDCF-mediated responses remains unclear. We tested the hypothesis that ER stress plays a role in EDCF-mediated responses via activation of the cPLA2/COX pathway in the aorta of the spontaneously hypertensive rat (SHR). Male SHR and Wistar-Kyoto rats (WKY) were treated with ER stress inhibitor, tauroursodeoxycholic acid or 4-phenlybutyric acid (TUDCA or PBA, respectively, 100 mg·kg−1·day−1 ip) or PBS (control, 300 μl/day ip) for 1 wk. There was a decrease in systolic blood pressure in SHR treated with TUDCA or PBA compared with control SHR (176 ± 3 or 181 ± 5, respectively vs. 200 ± 2 mmHg). In the SHR, treatment with TUDCA or PBA normalized aortic (vs. control SHR) 1) contractions to acetylcholine (ACh), AA, and tert-butyl hydroperoxide, 2) ACh-stimulated releases of prostanoids (thromboxane A2, PGF2α, and prostacyclin), 3) expression of COX-1, 4) phosphorylation of cPLA2 and ERK1/2, and 5) production of H2O2. Our findings demonstrate a novel interplay between ER stress and EDCF-mediated responses in the aorta of the SHR. Moreover, ER stress inhibition normalizes such responses by suppressing the cPLA2/COX pathway.

Download Full-text

Norepinephrine levels in paraventricular nucleus of spontaneously hypertensive rats: role of neuropeptide Y

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1993.265.3.h893 ◽

1993 ◽

Vol 265 (3) ◽

pp. H893-H898 ◽

Cited By ~ 4

Author(s):

N. D. Woo ◽

K. Mukherjee ◽

P. K. Ganguly

Keyword(s):

Neuropeptide Y ◽

Paraventricular Nucleus ◽

Spontaneously Hypertensive Rats ◽

Hypertensive Rats ◽

Spontaneously Hypertensive ◽

Central Neurons ◽

Sympathetic Regulation ◽

Series Of Experiments ◽

Wistar Kyoto

Recent evidence supports the view that the sympathetic system actively participates in the development of hypertension. Because norepinephrine, contained within central neurons involved in cardiovascular sympathetic regulation, is known to coexist with neuropeptide Y, it is possible that a functional interaction between neuropeptide Y and norepinephrine exists within the brain. In an effort to clarify whether or not central catecholamine systems are modulated by neuropeptide Y in hypertensive situations, the paraventricular nucleus of spontaneously hypertensive rats was exposed to neuropeptide Y (10(-9) M), and levels of norepinephrine were sampled by microdialysis. Norepinephrine levels in spontaneously hypertensive rats were significantly increased and did not change after exposure to neuropeptide Y, in sharp contrast to the decreases seen in Wistar-Kyoto controls. To ascertain whether these alterations in norepinephrine control were specific to the model used, a similar series of experiments was carried out in the paraventricular nucleus of aortic-banded rats. These studies supported the previous findings. Norepinephrine levels in aortic-banded rats were markedly elevated when compared with sham-operated controls and demonstrated no change after exposure to neuropeptide Y, whereas decreases of > 50% were seen in sham-operated controls. These results support the view that mechanisms normally involving neuropeptide Y as a neuromodulator in the paraventricular nucleus are altered in hypertensive situations. It is suggested that hypertension may precipitate changes in mechanisms involving brain neuropeptide Y and increased sympathetic activity.

Download Full-text

ADRENALINE (ADR) INTERACTIONS WITH THROMBIN AND COLLAGEN -EFFECTS ON PLATELET ARACHIDONATE RELEASE AND 5HT SECRETION AND ROLE OF ENDOGENOUSLY FORMED THROMBOXANE A2 (TxA2)

10.1055/s-0038-1643384 ◽

1987 ◽

Author(s):

Y Patel ◽

S Krishnamurthi ◽

V V Kakkar

Keyword(s):

Arachidonic Acid ◽

Platelet Aggregation ◽

Adenylate Cyclase ◽

Synergistic Effects ◽

Thromboxane A2 ◽

Human Platelets ◽

Presence And Absence ◽

Pre Treatment ◽

Arachidonate Release

We have examined the effect of combinations of ADR + thrombin (T) and ADR + collagen (C) on platelet arachidonate release and 5HT secretion, and assessed the role of endogenously formed TxA2 on these responses using indomethacin (I). Washed, human platelets prelabelled with [3H]-arachidonic acid (AA) or [14C]-5HT were used, ADR was added 10 sec before T or C and the reaction was terminated 3 min later. In the range 1-100μM, ADR induced no detectable aggregation or 5HT secretion but potentiated platelet aggregation when added with sub-threshold concentrations of T or C, which on their own induced no aggregation. At 2-4 fold higher concentrations of T and C (threshold for 5HT secretion), 5HT secretion and AA/TXB2 release were also potentiated by ADR (1-10μM) by 30-50%. Pre-treatment of platelets with I (10μM) abolished threshold T and C-induced 5HT secretion, as well as its potentiation by ADR. However, approximately 2-fold and 5-fold higher concentrations of T and C respectively were able to induce 'I-insensitive'secretion, which was further potentiated by ADR. In I-treated platelets, C-induced AA release and its potentiation by ADR were also abolished suggesting a role for endogenously formed TxA2 This was confirmed by addition of the TxA2 mimetic, U46619 (0.3μM), which potentiated C-induced AA release in the presence and absence of ADR, even though it induced no AA release on its own or, in combination with ADR alone in the absence of collagen. The latter suggests agonist specificity regarding the ability of TxA2 to synergistically stimulate AA release. Finally, unstirred platelets in PRP pre-incubated with ADR (10μM) for 120 min lost their responsiveness to ADR, when eventually stirred; however, these 'ADR-desensitised' platelets when washed and resuspended, were able to demonstrate synergistic effects on secretion when stimulated with ADR+T or ADR+C. This is analogous to the previously demonstrated ability of ADR to inhibit adenylate cyclase even in 'ADR-desensitised' platelets and re-inforces the separation regarding the mechanisms underlying the various effects of ADR on platelets.

Download Full-text

Role of cholinergic receptors in adrenal catecholamine secretion in spontaneously hypertensive rats

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1999.277.4.r1057 ◽

1999 ◽

Vol 277 (4) ◽

pp. R1057-R1062 ◽

Cited By ~ 2

Author(s):

Takahiro Nagayama ◽

Takayuki Matsumoto ◽

Makoto Yoshida ◽

Mizue Suzuki-Kusaba ◽

Hiroaki Hisa ◽

...

Keyword(s):

Electrical Stimulation ◽

Muscarinic Receptors ◽

Nicotinic Receptors ◽

Spontaneously Hypertensive Rats ◽

Adrenal Glands ◽

Hypertensive Rats ◽

Spontaneously Hypertensive ◽

Wky Rats ◽

Wistar Kyoto

We investigated the role of nicotinic and muscarinic receptors in secretion of catecholamines induced by transmural electrical stimulation (ES) from isolated perfused adrenal glands of spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats. ES (1–10 Hz) produced frequency-dependent increases in epinephrine (Epi) and norepinephrine (NE) output as measured in perfusate. The ES-induced increases in NE output, but not Epi output, were significantly greater in adrenal glands of SHRs than in those of WKY rats. Hexamethonium (10–100 μM) markedly inhibited the ES-induced increases in Epi and NE output from adrenal glands of SHRs and WKY rats. Atropine (0.3–3 μM) inhibited the ES-induced increases in Epi and NE output from adrenal glands of SHRs, but not from those of WKY rats. These results suggest that endogenous acetylcholine-induced secretion of adrenal catecholamines is predominantly mediated by nicotinic receptors in SHRs and WKY rats and that the contribution of muscarinic receptors may be different between these two strains.

Download Full-text