Strengthening Non-Communicable Disease Research Capacity in Thailand: Leveraging PhD Nurses and other Health Professionals.

The mounting burden of non-communicable disease (NCD) in lower-to-middle income countries (LMIC) was recognized two decades ago by the World Health Organization (WHO). In 2012, the WHO reported that NCDs were responsible for 71% of total deaths in Thailand. Thus, the goal of our ongoing NIH D43 training grant is to increase the quantity and quality of NCD research in Thailand by producing nurse scientists with the capacity to design, implement, guide, and evaluate cutting-edge research projects. The three components of the program are 1) a long-term component to train 10 Thai postdoctoral scholars that includes one year of intensive mentored research training in the US and one year implementing a research project back in Thailand; 2) a short-term component that provides an opportunity for Thai scientists, teachers, administrators and policy makers to take classes in areas relevant to NCD; 3) a yearly two-day NCD workshop to be held in different regions of Thailand. To date, 8 long-term trainees have completed various stages of their training. Eleven short-term trainees have taken courses at the University of Michigan. Two NCD workshops have been held so far under the auspices of the grant with a third planned for 2018. The ability to participate in the in-depth activities provided by the grant have not only expanded knowledge around NCD's, but also has enabled US and Thai investigators to grow in the area of culturally informed research.

Intracellular ATP can regulate afferent arteriolar tone via ATP-sensitive K+ channels in the rabbit.

Studies were performed to assess whether ATP-sensitive K+ (KATP) channels on rabbit preglomerular vessels can influence afferent arteriolar (AA) tone. K+ channels with a slope conductance of 258 +/- 13 (n = 7) pS and pronounced voltage dependence were demonstrated in excised patches from vascular smooth muscle cells of microdissected preglomerular segments. Channel activity was markedly reduced by 1 mM ATP and in a dose-dependent fashion by glibenclamide (10(-9) M to 10(-6) M), a specific antagonist of KATP channels. 10(-5) M diazoxide, a K+ channel opener, activated these channels in the presence of ATP, and this effect was also blocked by glibenclamide. To determine the role of these KATP channels in the control of vascular tone, diazoxide was tested on isolated perfused AA. After preconstriction from a control diameter of 13.1 +/- 1.1 to 3.5 +/- 2.1 microns with phenylephrine (PE), addition of 10(-5) M diazoxide dilated vessels to 11.2 +/- 0.7 microns, which was not different from control. Further addition of 10(-5) M glibenclamide reconstricted the vessels to 5.8 +/- 1.5 microns (n = 5; P less than 0.03). In support of its specificity for KATP channels, glibenclamide did not reverse verapamil induced dilation in a separate series of experiments. To determine whether intracellular ATP levels can effect AA tone, studies were conducted to test the effect of the glycolytic inhibitor 2-deoxy-D-glucose. After preconstriction from 13.4 +/- 3.2 to 7.7 +/- 1.3 microns with PE, bath glucose was replaced with 6 mM 2-deoxy-D-glucose. Within 10 min, the arteriole dilated to a mean value of 11.8 +/- 1.4 microns (n = 6; NS compared to control). Subsequent addition of 10(-5) M glibenclamide significantly reconstricted the vessels to a diameter of 8.6 +/- 0.5 micron (P less than 0.04). These data demonstrate that KATP channels are present on the preglomerular vasculature and that changes in intracellular ATP can directly influence afferent arteriolar tone via these channels.

Calcium-related abnormalities in lymphocytes from genetically hypertensive rats.

We examined the effect of various external calcium concentrations on net potassium efflux and net sodium influx in lymphocytes from spontaneously hypertensive rats (SHR), stroke-prone spontaneously hypertensive rats (SHRSP), and Wistar-Kyoto rats (WKY). Net potassium efflux was greater in lymphocytes from SHRSP than in those from WKY at external calcium concentrations of 0.1, 0.3, 1.0, and 3.0 mM but not at 0 mM (14.9 +/- 0.8 vs 13.0 +/- 0.7 mmol per kilogram of dry weight per hour, respectively). Net sodium influx in lymphocytes from SHRSP was greater than in those from WKY at all external calcium concentrations tested (0, 0.1, 1.0, and 3.0 mM). In contrast to lymphocytes from WKY, net potassium efflux and net sodium influx in lymphocytes from SHRSP were not significantly higher at 0 than at 0.1 mM external calcium concentration. Lymphocytes from SHRSP had elevated intracellular free calcium concentrations (173.6 +/- 7.4 nM, n = 8), as compared with lymphocytes from WKY (98.1 +/- 9.1 nM, n = 8). These data suggest that the interaction of calcium with the lymphocyte plasma membrane directly affects monovalent ion permeability and is altered in lymphocytes from SHRSP, as compared with those from WKY. Our findings support the hypothesis that in hypertension there is a generalized increase in cell membrane permeability to calcium and monovalent ions, which may result from a reduced number of calcium-binding sites on the plasma membrane.

Calcium sensitivity of Ca2(+)-activated K+ channels in spontaneously hypertensive stroke-prone rats.

In previous studies, we measured a greater intracellular free calcium concentration and net potassium efflux, possibly calcium activated, in lymphocytes from spontaneously hypertensive stroke-prone rats (SHRSP) as compared with Wistar-Kyoto (WKY) rats. In this study, we addressed two related questions: 1) Can the greater intralymphocytic calcium concentration of the SHRSP account for the greater net potassium efflux? 2) Is the calcium sensitivity of calcium-activated potassium channels in lymphocytes from SHRSP different as compared with that of those from WKY rats? Ionomycin, a calcium ionophore, caused a concentration-dependent and proportional increase in net potassium efflux and intracellular free calcium concentration in lymphocytes from both strains of rat. Based on the relations between net potassium efflux and intracellular free calcium concentration established with ionomycin, the resting net potassium efflux of lymphocytes from SHRSP is greater than would be predicted based on the resting intracellular free calcium concentration. Using the patch clamp technique, we were able to identify and characterize a calcium-activated potassium channel in the plasma membrane of lymphocytes from both strains of rat. Potassium currents were recorded that had a slope conductance of 18.1 +/- 1.49, n = 6, and 18.5 +/- 1.44, n = 7, in WKY rat and SHRSP thymocytes, respectively. The channel exhibited rectification of the outward current in both strains of rat. Channels tended to appear in clusters of two or more per patch and were recorded in 30-50% of the patches examined. Calcium sensitivity of the channels was similar; maximum activation occurred at 700 nM free calcium concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Potassium channels and vascular reactivity in genetically hypertensive rats.

In hypertension, membrane potassium permeability and vascular reactivity are increased. This study characterizes a potassium-selective channel and contractions to barium, a potassium channel inhibitor, in vascular smooth muscle (tail artery) from spontaneously hypertensive stroke-prone rats (SHRSP) and normotensive Wistar-Kyoto (WKY) rats. Smooth muscle cells were isolated by enzymatic digestion, and potassium channel activity was characterized by using patch-clamp technique (inside-out configuration). Isometric contractile activity was evaluated in helically cut arterial strips by using standard muscle bath methodology. In membrane patches, a voltage-gated, calcium-insensitive, potassium-selective channel of large conductance (200 picosiemens) was observed. The channel did not conduct sodium or rubidium. Barium (10(-6) to 10(-4) M) produced a dose-dependent blockade of channel activity. These channel characteristics did not differ in SHRSP and WKY rat cells. After treatment with 35 mM KCl, barium (10(-5) to 10(-3) M) caused greater contractions in SHRSP arteries compared with arteries in WKY rats. The contractions to barium were markedly attenuated in calcium-free solution, and nifedipine and verapamil abolished contractions induced by barium in depolarizing solution. We conclude that increased vascular reactivity to barium in SHRSP arteries is not due to an alteration in the biophysical properties of the potassium channel studied.

Lymphocyte abnormalities in three types of hypertension in the rat.

Lymphocyte number and weight and their sodium and potassium contents and net passive fluxes were measured in spontaneously hypertensive stroke-prone rats, deoxycorticosterone acetate-treated rats, and two-kidney, one clip renal hypertensive rats. Wistar-Kyoto rats were used as controls for the spontaneously hypertensive stroke-prone rats, and normal intact Sprague-Dawley rats were used as controls for the others. Blood lymphocyte count was higher and lymphocyte weight was lower in the hypertensive rats. Intralymphocytic sodium content (millimoles per kilogram of dry weight) was elevated in the three forms of hypertension as compared with control values (spontaneously hypertensive stroke-prone rats, 43.0 +/- 1.7 vs Wistar-Kyoto rats, 37.3 +/- 1.3; deoxycorticosterone acetate-treated rats, 44.4 +/- 3.1 vs Sprague-Dawley rats, 36.1 +/- 1.7; one-kidney, one clip rats, 50.5 +/- 3.7 vs Sprague-Dawley rats, 38.9 +/- 2.0). Intralymphocytic potassium content was not significantly altered in any of the forms of hypertension. Lymphocytes from spontaneously hypertensive stroke-prone rats and deoxycorticosterone acetate-treated rats exhibited elevated net sodium fluxes (millimoles per kilogram of dry weight per hour) as compared with those of controls (spontaneously hypertensive stroke-prone rats, 7.00 +/- 0.99 vs Wistar-Kyoto rats, 4.89 +/- 0.63; deoxycorticosterone acetate-treated rats, 7.58 +/- 0.97 vs Sprague-Dawley rats, 5.6 +/- 0.64). Net potassium fluxes were significantly elevated only in the spontaneously hypertensive stroke-prone rats (14.07 +/- 1.70 vs 8.23 +/- 1.04 in Wistar-Kyoto rats). Sodium and potassium fluxes in lymphocytes from two-kidney, one clip rats and Sprague-Dawley rats were not significantly different.(ABSTRACT TRUNCATED AT 250 WORDS)

Dietary calcium and cell membrane abnormality in genetic hypertension.

Adult stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto (WKY) rats were maintained for 10 weeks on one of two diets: 1.0% calcium content and 2.5% calcium content. At the end of this time rats were anesthetized, and blood pressure was determined by means of aortic cannulation; then the rats were exsanguinated. Lymphocytes were isolated for determination of intracellular sodium and potassium concentrations, net sodium influx, net potassium efflux, and intracellular free calcium concentration. Serum ionized calcium was also measured. The increase in calcium content of their diet had no effect on intracellular sodium and potassium concentrations in lymphocytes from WKY rats and SHRSP. In lymphocytes from WKY rats, none of the parameters was affected by the change in dietary calcium intake. In contrast, in lymphocytes from SHRSP the increase in dietary calcium from 1.0 to 2.5% led to significant decreases in net potassium efflux (13.3 +/- 2.3 vs. 19.7 +/- 1.4 mmol/kg dry wt/hr, p less than 0.05, analysis of variance), intracellular free calcium concentration (114.5 +/- 10.2 vs. 166.2 +/- 11.2 nM, p less than 0.001), and systolic blood pressure (125.3 +/- 13.6 vs. 183.3 +/- 16.6 mm Hg, p less than 0.01). Serum ionized calcium increased in SHRSP (2.40 +/- 0.04 vs. 2.16 +/- 0.03, p less than 0.01) but not in WKY rats (2.34 +/- 0.05 vs. 2.31 +/- 0.05) fed the high calcium diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium current in smooth muscle cells from normotensive and genetically hypertensive rats.

Genetic hypertension results from numerous phenotypic expressions. We hypothesized that increased calcium current in vascular smooth muscle of genetically hypertensive animals is partly responsible for observed increases in agonist sensitivity, contractility, and calcium influx. Using adult, spontaneously hypertensive stroke-prone rats (SHRSP) and normotensive Wistar-Kyoto (WKY) controls from an inbred colony, we characterized calcium current in smooth muscle cells isolated from cerebral arteries. Calcium current in WKY cells reached a maximum of -27.7 +/- 2.7 pA (n = 32) at +20 mV. Peak inward current at +20 mV in SHRSP cells had a mean amplitude of -44.4 +/- 3.0 pA (n = 72, P < .05). SHRSP cells exhibited a higher calcium current density. Maximal inward current normalized to cell capacitance yielded mean values of 2.07 +/- 0.11 pA/pF for WKY (n = 32) and 2.80 +/- 0.12 pA/pF (n = 79) for SHRSP (P < .05) cells. Transient-type Ca2+ channel current had the same magnitude and current-voltage relation in both cell types, giving an L-type/T-type ratio of 3.85 for WKY and 6.25 for SHRSP cells. The voltage-dependent inactivation curve for SHRSP calcium current was shifted to the right only over the range of -50 to -30 mV, but the half-maximal inactivation voltages and Boltzmann coefficients were not significantly different between cell types. Increased calcium inward current in this model of genetic hypertension could account in part for altered calcium homeostasis and increased vascular reactivity, contributing to hypertension and vasospasm.

Decreased ATP sensitivity of a K+ channel and enhanced vascular smooth muscle relaxation in genetically hypertensive rats.

OBJECTIVE: To characterize an ATP-sensitive K+ channel at the single-channel and tissue level in the vascular smooth muscle of normotensive and genetically hypertensive rats. METHODS: Age- and sex-matched Wistar-Kyoto (WKY) rats and stroke-prone spontaneously hypertensive rats (SHRSP) were used. Patch-clamp single-channel recording was used to measure K+ channel activity in dissociated tail artery cells. The effect of the K+ channel-opener diazoxide and the specific ATP-sensitive K+ channel antagonist glyburide on isometric force development in isolated tail artery strips was determined by a standard muscle bath technique. RESULTS: The concentration of ATP that caused half-maximal reduction in channel activity was greater in the SHRSP than in the WKY rats. Tail artery strips and cells from SHRSP were more sensitive to the effect of diazoxide on relaxation and channel activity, and less responsive to the effect of glyburide, than were those from WKY rats. CONCLUSIONS: The decreased ATP sensitivity of this K+ channel may partly compensate for the increased vascular reactivity in hypertension, and the change in this property of the channel may be responsible for the altered sensitivity to diazoxide and glyburide in SHRSP.

Genetic relationship between a lymphocyte membrane abnormality and blood pressure in spontaneously hypertensive stroke prone and Wistar-Kyoto rats.

The fluxes of sodium and potassium across the lymphocyte membrane in spontaneously hypertensive, stroke prone rats (SHRSP) are greater than those in Wistar-Kyoto rats (WKY). In order to define the relationship of these abnormal membrane traits to arterial pressure elevation, their distributions were studied in F1, F2, and backcross progeny of the cross of SHRSP and WKY rats. In the F1 generation, arterial pressure and potassium efflux values resembled those of the WKY parents suggesting that these traits are dominant in the normotensive rat; sodium influx values resembled those of the SHRSP parents. There was poor correlation (r = 0.45) between blood pressure and sodium influx in the F2 generation. There was a high correlation between blood pressure and potassium efflux (r = 0.86), suggesting that these two traits may be under the control of a common genetic mechanism.

WAY 120,491 activates ATP-sensitive potassium channels in rat tail artery.

WAY 120,491 lowers blood pressure in a number of animal models. Its mechanism of action appears to be the selective opening of K+ channels in vascular smooth muscle cells. The patch clamp technique was used to determine the specific K+ channel affected by WAY 120,491 in isolated tail artery cells of the rat. In the inside-out configuration, WAY 120,491 caused a dose dependent partial reversal of the ATP-induced inactivation of channel activity. Glyburide (10 microM) almost completely inhibited the effect of WAY 120,491. WAY 120,491 had no effect on Ca-activated K+ channels inactivated by the absence of Ca2+. In the whole cell configuration, 10 microM WAY 120,491 increased outward K+ current. 10 microM glyburide completely reversed the increase in current. WAY 120,491 appears to be a relatively specific activator of ATP-sensitive K+ channels in vascular smooth muscle.

Many membrane abnormalities in hypertension result from one primary defect.

Evidence has been presented that: 1.) Changes in lipid bilayer alter the function of integral membrane proteins. 2.) There is less calcium bound to the plasma membrane in hypertension. 3.) Structural and functional abnormalities of the lipid bilayer have been reported in genetic hypertension. We hypothesize that multiple abnormalities of membrane transport systems in hypertension are secondary to an inherent abnormality of the lipid bilayer in which these transport proteins reside.

Effects of felodipine on blood pressure and lymphocyte membrane characteristics in spontaneously hypertensive stroke-prone rats.

Adult spontaneously hypertensive stroke-prone rats (SHRSP) and age- and sex-matched Wistar-Kyoto rats (WKY) were treated with the calcium-channel antagonist felodipine for 2-4 weeks (1 mg/g in rat chow powder). Control rats of both strains were fed untreated chow. At the end of the treatment period we measured blood pressure, net potassium efflux and intralymphocytic calcium concentration. In the untreated rats the values for these parameters were significantly higher in SHRSP than in WKY. Felodipine treatment caused a reduction in the SHRSP values to WKY levels. We conclude that the high blood pressure of SHRSP results from an increase in membrane permeability to calcium. The resultant increase in intralymphocytic calcium concentration causes an increase in net potassium efflux via calcium-activated potassium channels. By reducing the membrane permeability to calcium with felodipine, these parameters were returned to normal or near-normal levels.

Cooling-induced contraction and protein tyrosine kinase activity of isolated arterioles in secondary Raynaud's phenomenon.

OBJECTIVE: To investigate the response of skin arterioles from control subjects and patients with scleroderma and Raynaud's phenomenon (RP/SSc) to cooling and modulators of protein tyrosine kinase (PTK) activity. METHODS: We used the microvessel perfusion technique to characterize the response of isolated dermal arterioles (100-200 microm, outside diameter) from normal (n = 17) and RP/SSc (n = 17) subjects to cooling from 37 degrees to 31 degrees C. Fluorescent immunohistochemistry was used to measure tyrosine phosphorylation. RESULTS: Arterioles from control subjects exhibited dilation in response to cooling from 37 to 31 degrees C whereas those from RP/SSc subjects contracted (+4.3 +/- 1.7 vs -16.7 +/- 3.1%, P < 0.05, n = 6). In the presence of the protein tyrosine phosphatase inhibitor sodium orthovanadate (SOV, 10 microM), the response of arterioles from control subjects did not change; however, arterioles from RP/SSC subjects exhibited a significantly greater contraction (-72.6 +/- 19.7%; P < 0.05, n = 6). Tyrosine phosphorylation of arterioles at 37 degrees C from control and RP/SSc subjects was similar. In response to cooling to 31 degrees C, however, arterioles from RP/SSc subjects exhibited a significantly greater increase in tyrosine phosphorylation compared with those from control subjects (43 +/- 7.0% vs 10 +/- 3.8%; P < 0.01). SOV increased tyrosine phosphorylation in arterioles from both groups (73 +/- 11.6% vs 42 +/- 5.6%; P < 0.05, n = 5). Arterioles from RP/SSC subjects precontracted with norepinephrine exhibited greatly attenuated relaxation to acetylcholine compared with those from control subjects. CONCLUSION: The results of this study support the view that the hallmark of Raynaud's phenomenon associated with scleroderma, cooling-induced vasospasm, appears to be mediated by an increase in PTK activity possibly exacerbated by impaired endothelium-dependent vasodilation.

Cell membrane permeability in hypertension.

A great deal of evidence has been presented which suggests that a generalized increase in cell membrane permeability to various ions is a fundamental defect associated with hypertension. In addition, some of the evidence is supportive of a central role of this defect in the pathogenesis of hypertension. The source of this defect may lie in the reduced calcium binding capacity of the cell membrane. Because of this reduced capacity the membrane is less stable and is more permeable to various ions.

Effect of modulators of protein tyrosine kinase activity on gender-related differences in vascular reactivity at reduced temperature.

We used the isolated-muscle-bath technique to examine the effect of protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) inhibitors on the response of rings of tail artery from male and female rats to cooling and reduced temperature in the absence and presence of two PTK-dependent (clonidine and serotonin) and one PTK-independent (phenylephrine, PE) agonists. At 37 degrees C, reactivity to clonidine, serotonin, and PE was the same in tail artery from female and male rats. At 25 degrees C, reactivity to clonidine and serotonin, but not PE, was greater in tail artery from female rats compared with those from male rats. Sodium orthovanadate (SOV) eliminated the gender-related difference in the contractile effect of clonidine and serotonin at 25 degrees C. The sensitivity to relaxation by genistein was considerably greater for clonidine and serotonin at both temperatures as compared with PE. At 25 degrees C the sensitivity to genistein was greater for the clonidine and serotonin-contracted rings from female rats. In the presence of SOV, temperature reduction led to contraction of rat-tail artery. This effect was greater in rings from female rats. Our results strongly implicate differences in the activity of the PTK transduction pathway as the cause of the observed gender-related differences in agonist-mediated contraction at 25 degrees C and in cold-induced vasoconstriction.

Effect of temperature and modulators of protein tyrosine kinase activity on the reactivity of isolated venules in secondary Raynaud's phenomenon.

OBJECTIVE: To investigate the response of skin venules from healthy controls and scleroderma patients with Raynaud's phenomenon (RP/SSc) to cooling and to modulators of protein tyrosine kinase (PTK) activity at normal and reduced temperature. METHODS: We used the microvessel perfusion technique to characterize the response of isolated dermal venules (200-400 microm outside diameter) from normal (n = 10) and RP/SSc (n = 8) subjects to cooling and to contractile agents at 37 and 31 degrees C. RESULTS. The response to clonidine at 37 degrees C was less in venules from patients with RP/SSc compared to controls; the contraction to serotonin was greater in venules from RP/SSc patients versus controls; at 31 degrees C, venules from RP/SSc patients contracted to both clonidine and serotonin to a greater extent versus controls; and contraction to these agonists was reversed by cumulative addition of genistein (1-100 microM). Venules from controls and patients with RP/SSc exhibited slight vasodilation to cooling from 37 to 31 degrees C. In the presence of the protein tyrosine phosphatase inhibitor sodium orthovanadate (10 microM), venules from controls now exhibited a small contraction (-5.1 +/- 3.2%) and venules from RP/SSC subjects a significantly greater contraction (-38.7 +/- 9.0%; p < 0.05). CONCLUSION: Our study supports the view that RP/SSc is the result of defects in the peripheral vasculature.

Cardiac actions of bovine parathyroid hormone fragment (1-34) in some lower vertebrates.

Only recently have the cardiac actions of parathyroid hormone and its N-terminal (1-34) fragment (bPTH(1-34] been examined. Parathyroid hormone was found to exert a positive chronotropic effect on the heart tissue of several mammals and one amphibian in vivo and/or in vitro. The purpose of the present study was to examine in vitro the heart tissue of several lower vertebrates, particularly aquatic vertebrates, for responsiveness to bPTH(1-34) and, for comparative purposes, isoproterenol. Heart tissue (atrium or the entire heart) from all the animals studied (trout, mudpuppy, bullfrog tadpole, and adult bullfrog) responded to isoproterenol in a dose-dependent fashion with increased heart rate and contractile force. Only the atria from the adult bullfrog, however, responded in a similar manner to the administration of bPTH(1-34). In all cases propranalol (10(-6) M) was able to block both chronotropic and inotropic effects of isoproterenol but had no effect on the cardiac stimulation induced by bPTH(1-34) in the adult bullfrog atrium. The data suggests that cardiac responsiveness to bPTH(1-34) is associated with a terrestrial as opposed to an aquatic existence.

Calcium and vascular smooth muscle membrane in hypertension.

A major focus of past and recent research in hypertension has been on the characterization of the nature of the vasculature changes which lead to the observed increase in total peripheral resistance responsible for the elevation of arterial pressure. Here we survey recent evidence which suggests that altered handling of calcium is a primary membrane defect in hypertension. Evidence for the primacy of this defect is provided by studies demonstrating a reduced ability of the membrane to bind calcium in diverse tissues from hypertensive animals. The reduced calcium binding ability appears to be responsible for a greater membrane permeability to monovalent and divalent cations. This greater permeability contributes to the increased sensitivity to vasoconstrictor stimuli of vascular smooth muscle in hypertension.