HDL associates with insulin resistance and beta-cell dysfunction in South Asian families at risk of type 2 diabetes.

OBJECTIVE: Dyslipidemia precedes type 2 diabetes (T2D) and worsens with increasing glucose intolerance. First degree relatives of T2D patients have an increased risk to develop dyslipidemia and glucose intolerance. The aim of the present study was to assess the relation between the development of dyslipidemia and glucose intolerance in first-degree relatives of T2D patients. RESEARCH DESIGN AND METHODS: Fasting lipoprotein profiles were determined by density gradient ultracentrifugation in T2D patients and their first-degree relatives (42 Caucasians and 33 South Asians), and in 29 normoglycemic controls from non-T2D families. Glucose tolerance, insulin sensitivity index (ISI) and insulin disposition index (DI) were assessed by an extended, frequently sampled oral glucose tolerance test (OGTT), and fractional insulin synthesis rate (FSR) was measured by 13C-leucine enrichment in urinary C-peptide during the OGTT. RESULTS: Of the first-degree relatives, 40, 16 and 19 had NGT, prediabetes and T2D, respectively. NGT family members had lower plasma HDL-cholesterol (HDLC) (1.34 ± 0.07 vs 1.58 ± 0.06 mmol/L; p = 0.015), HDL2-C (0.41 ± 0.05 vs 0.57 ± 0.05 mmol/L; p = 0.021) and HDL3-C (0.62 ± 0.03 vs 0.72 ± 0.02 mmol/L; p = 0.043) than controls. HDL2-C levels tended to decrease with increasing glucose intolerance state. In South Asians, buoyant LDL-C levels decreased with increasing glucose intolerance state (p = 0.006). In South Asian families, HDL-C correlated with both ISI and DI (ß 0.42; p = 0.04 and ß 0.53; p = 0.01, respectively), whereas HDL2-C and HDL3-C levels correlated with DI (ß 0.64; p = 0.002 and ß 0.57; p = 0.005, respectively). HDL2-C and plasma triglyceride correlated with FSR (ß 0.48; p = 0.033 and ß -0.50; p = 0.029, respectively). CONCLUSIONS: Low HDL2-C and HDL3-C levels are present in NGT first-degree relatives of T2D patients, and HDL2-C tend to decrease further with increasing glucose intolerance. In South Asian families HDL2-C and HDL3-C levels linked predominantly to deteriorating beta cell function.

Breakfast partly restores the anti-inflammatory function of high-density lipoproteins from patients with type 2 diabetes mellitus.

Background and aims: High-density lipoproteins (HDL) of patients with type 2 diabetes mellitus (T2DM) have impaired anti-inflammatory activities. The anti-inflammatory activity of HDL has been determined ex vivo after isolation by different methods from blood mostly obtained after overnight fasting. We first determined the effect of the HDL isolation method, and subsequently the effect of food intake on the anti-inflammatory function of HDL from T2DM patients. Methods: Blood was collected from healthy controls and T2DM patients after an overnight fast, and from T2DM patients 3 h after breakfast (n = 17 each). HDL was isolated by a two-step density gradient ultracentrifugation in iodixanol (HDLDGUC2), by sequential salt density flotation (HDLSEQ) or by PEG precipitation (HDLPEG). The anti-inflammatory function of HDL was determined by the reduction of the TNFα-induced expression of VCAM-1 in human coronary artery endothelial cells (HCAEC) and retinal endothelial cells (REC). Results: HDL isolated by the three different methods from healthy controls inhibited TNFα-induced VCAM-1 expression in HCAEC. With apoA-I at 0.7 µM, HDLDGUC2 and HDLSEQ were similarly effective (16% versus 14% reduction; n = 3; p > 0.05) but less effective than HDLPEG (28%, p < 0.05). Since ultracentrifugation removes most of the unbound plasma proteins, we used HDLDGUC2 for further experiments. With apoA-I at 3.2 µM, HDL from fasting healthy controls and T2DM patients reduced TNFα-induced VCAM-1 expression in HCAEC by 58 ± 13% and 51 ± 20%, respectively (p = 0.35), and in REC by 42 ± 13% and 25 ± 18%, respectively (p < 0.05). Compared to preprandial HDL, postprandial HDL from T2DM patients reduced VCAM-1 expression by 56 ± 16% (paired test: p < 0.001) in HCAEC and by 34 ± 13% (paired test: p < 0.05) in REC. Conclusions: The ex vivo anti-inflammatory activity of HDL is affected by the HDL isolation method. Two-step ultracentrifugation in an iodixanol gradient is a suitable method for HDL isolation when testing HDL anti-inflammatory function. The anti-inflammatory activity of HDL from overnight fasted T2DM patients is significantly impaired in REC but not in HCAEC. The anti-inflammatory function of HDL is partly restored by food intake.

Role of plasma adiponectin on the HDL-cholesterol raising effect of atorvastatin in patients with type 2 diabetes.

OBJECTIVE: Adiponectin, secreted by adipose tissue, plays an important role in lipoprotein metabolism and also affects carbohydrate and insulin pathways. We studied the effects of atorvastatin treatment on plasma adiponectin and high density cholesterol (HDL) levels in patients with type 2 diabetes. RESEARCH DESIGN AND METHODS: In the 'Diabetes Atorvastatin Lipid Intervention' (DALI) study, a randomized placebo-controlled study on the effects of atorvastatin treatment in 194 patients with type 2 diabetes and mildly elevated plasma triglycerides, adiponectin levels, lipoproteins, cholesteryl ester transfer protein (CETP) mass, as well as postheparin lipoprotein lipase (LPL) and hepatic lipase (HL) activities were assessed at baseline and after 6 months of treatment (placebo, 10 mg or 80 mg atorvastatin). RESULTS: At baseline, plasma adiponectin levels were positively associated with HDL cholesterol (r = 0.40, p < 0.001), and apoA-I (r = 0.38, p < 0.001) in both males and females. Adiponectin was negatively associated with triglycerides (r = -0.26, p < 0.001) in males as well as in females. Atorvastatin treatment had no effect on plasma adiponectin levels. However, adiponectin levels at baseline significantly predicted the effect of atorvastatin treatment on HDL-cholesterol (p = 0.007), i.e. patients with the highest baseline plasma adiponectin concentration (tertile 3) displayed the largest increase in plasma HDL cholesterol during treatment (8-10%), while the HDL-cholesterol increase in tertile 1 was almost negligible (1-3%). CONCLUSION: In this study, high baseline plasma adiponectin levels significantly affect the HDL-cholesterol response to atorvastatin treatment in patients with type 2 diabetes and therefore may play a role in defining future treatment strategy.

Reduced leucocyte cholesteryl ester transfer protein expression in acute coronary syndromes.

OBJECTIVE: Cholesterol ester transfer protein (CETP) plays an important role in HDL cholesterol metabolism. Leucocytes, including monocyte-derived macrophages in the arterial wall synthesize and secrete CETP, but its role in atherosclerosis is unclear. The aim of the current study was to investigate the effect of acute coronary syndromes (ACS) on leucocyte CETP expression. RESEARCH DESIGN: Peripheral blood mononuclear cells (PBMCs) were freshly isolated from hospitalized ACS patients displaying Braunwald class IIIB unstable angina pectoris (UAP) on admission (t = 0) and at 180 days post inclusion (t = 180) for analysis of CETP expression. In addition, to prove the potential correlation between leucocyte CETP and ACS the effect of acute myocardial infarction on leucocyte CETP expression was studied in CETP transgenic mice. RESULTS: Upon admission, UAP patients displayed approximately 3-6 fold (P < 0.01) lower CETP mRNA and nearly absent CETP protein expression in PBMCs, as compared to healthy age-/sex-matched controls. Interestingly, CETP mRNA and protein levels were significantly elevated in PBMCs isolated from UAP patients (both stabilized and refractory) at t = 180 as compared to t = 0 (P < 0.01), which was correlated with a reduced inflammatory status after medical treatment. In agreement with the data obtained in UAP patients, markedly down-regulated leucocyte CETP mRNA expression was observed after coronary artery ligation in CETP transgenic mice, which also correlated with increased serum amyloid A levels. CONCLUSIONS: We are the first to report that episodes of UAP in humans and myocardial infarction in CETP transgenic mice are associated with reduced leucocyte CETP expression. We propose that the impairment in leucocyte CETP production is associated with an enhanced inflammatory status, which could be clinically relevant for the pathogenesis of ACS.

Appraisal of hepatic lipase and lipoprotein lipase activities in mice.

A variety of methods are currently used to analyze HL and LPL activities in mice. In search of a simple methodology, we analyzed mouse preheparin and postheparin plasma LPL and HL activities using specific polyclonal antibodies raised in rabbit against rat HL (anti-HL) and in goat against rat LPL (anti-LPL). As an alternative, we analyzed HL activity in the presence of 1 M NaCl, a condition known to inhibit LPL activity in humans. The assays were validated using plasma samples from wild-type and HL-deficient C57BL/6 mice. We now show that the use of 1 M NaCl for the inhibition of plasma LPL activity in mice may generate incorrect measurements of both LPL and HL activities. Our data indicate that HL can be measured directly, without heparin injection, in preheparin plasma, because virtually all HL is present in an unbound form circulating in plasma. In contrast, measurable LPL activity is present only in postheparin plasma. Both HL and LPL can be measured using the same assay conditions (low salt and the presence of apolipoprotein C-II as an LPL activator). Total lipase activity in postheparin plasma minus preheparin HL activity reflects LPL activity. Specific antibodies are not required.

Plasma apolipoprotein A5 and triglycerides in type 2 diabetes.

AIMS/HYPOTHESIS: Variation in the human apolipoprotein (APO) A5 gene (APOA5) is associated with elevated plasma triglycerides. However, data on the exact role of plasma concentrations of APOA5 in human triglyceride homeostasis are lacking. In the present study, we estimated plasma APOA5 levels in patients with type 2 diabetes at baseline and during atorvastatin treatment, a lipid-lowering treatment that results in a reduction in plasma triglycerides and APOC3. SUBJECTS, MATERIALS AND METHODS: Plasma APOA5 concentration was measured by ELISA in 215 subjects with type 2 diabetes, who were taken from the Diabetes Atorvastatin Lipid-lowering Intervention (DALI) study, a 30-week randomised, double-blind, placebo-controlled study, and given atorvastatin 10 mg or 80 mg daily. RESULTS: At baseline, average plasma APOA5 concentration was 25.7+/-15.6 mug/100 ml. Plasma APOA5 (R (s)=0.40), APOC3 (R (s)=0.72) and APOE (R (s)=0.45) were positively correlated with plasma triglyceride levels (all p<0.001). In multiple linear regression analysis, adjusted for age and sex, the variation in plasma triglycerides was explained mostly by APOC3 (52%) and only to a small extent by APOA5 (6%) and APOE (1%). Atorvastatin treatment decreased plasma triglycerides, APOA5, APOC3 and APOE (all p<0.0001). After treatment, APOC3 remained the major determinant of plasma triglyceride levels (59%), while the contributions of APOA5 and APOE were insignificant (2 and 3%). CONCLUSIONS/INTERPRETATION: Our findings reveal a positive association between plasma APOA5 and triglycerides in patients with type 2 diabetes. Treatment with atorvastatin decreased plasma APOA5, APOC3, APOE and triglycerides. In contrast to APOC3, APOA5 is not a major determinant of triglyceride metabolism in these patients.

Myocardial force development and structural changes associated with monocrotaline induced cardiac hypertrophy and heart failure.

In this study alterations are characterized which occur, in myocardial force development morphological appearance and protein composition, during the development of cardiac hypertrophy and heart failure in monocrotaline (MCT) treated rats. The transition from cardiac hypertrophy to heart failure was studied by comparing the results from control (CON) and two MCT groups (40 and 44 mg/kg body weight). The three experimental groups consisted of at least five animals each. Parameters studied were: body weight (measured daily), lung/body weight ratio, right ventricular wall volume and thickness, and force development in thin right ventricular trabeculae at 27 degrees C, using different extracellular calcium concentrations and pacing frequencies. MCT injection resulted in marked right ventricular hypertrophy and heart failure as evidenced by an up to 2-fold increase in lung/body weight ratio and a 1.7-fold increase in wall volume. The MCT groups showed a negative force-frequency relation and maximum force was up to 2-fold less than in the CON group. Protein analysis by means of one- and two-dimensional gel electrophoresis revealed a marked (7-fold) up-regulation of the slow myosin heavy chain isoform as well as a 4.5-fold increase in the content of the cytoskeletal protein desmin, whereas the mitochondrial protein ATP-synthase content was reduced. Hence MCT-induced cardiac hypertrophy and heart failure result in altered cellular calcium handling, depression of maximum force output, an increase in the economy of myocardial contraction and changes in cytoskeletal structure and energy supply.

The effect of a polynutrient supplement on fatigue and physical activity of patients with chronic fatigue syndrome: a double-blind randomized controlled trial.

BACKGROUND: The efficacy of dietary supplements in chronic fatigue syndrome (CFS) is uncertain, with conflicting evidence. AIM: To assess the effect of a polynutrient supplement on fatigue and physical activity of patients with CFS. DESIGN: Prospective randomized placebo-controlled, double-blind trial. METHODS: Fifty-three patients (16 males, 37 females) fulfilling the CDC criteria of CFS. The entry criteria were a score on the Checklist Individual Strength subscale fatigue severity (CIS fatigue) >or=40 and a weighted sum score of >or=750 for the eight subscales of the Sickness Impact Profile (SIP8) and no use of nutritional supplements in the 4 weeks prior to entry. The exclusion criteria were pregnancy and lactose intolerance. The intervention-a polynutrient supplement containing several vitamins, minerals and (co)enzymes, or placebo, twice daily for 10 weeks-was preceded by 2 weeks of baseline measurements. Outcome measurements took place in week 9 and 10 of the intervention. Five participants dropped out (4 supplement, 1 placebo). The main outcome measures were CIS fatigue score, number of CDC symptoms and SIP8 score. Efficacy analyses were performed on an intention-to-treat basis. RESULTS: No significant differences were found between the placebo and the treated group on any of the outcome measures: CIS fatigue +2.16 (95%CI -4.3 to +4.39, p=0.984); CDC symptoms +0.42 (95%CI -0.61 to +1.46, p=0.417); SIP8 +182 (95%CI -165 to +529, p=0.297). No patient reported full recovery. DISCUSSION: The findings do not support the use of a broad-spectrum nutritional supplement in treating CFS-related symptoms.

Atrial fibrillation in the goat induces changes in monophasic action potential and mRNA expression of ion channels involved in repolarization.

INTRODUCTION: Sustained atrial fibrillation (AF) is characterized by a marked shortening of the atrial effective refractory period (AERP) and a decrease or reversal of its physiologic adaptation to heart rate. The aim of the present study was to investigate whether the AF-induced changes in AERP in the goat are associated with changes in the atrial monophasic action potential (MAP) and whether an abnormal expression of specific ion channels underlies such changes. METHODS AND RESULTS: Following thoracotomy, MAPs were recorded from the free wall of the right atrium both before induction of AF (control) and after cardioversion of sustained AF (>2 months) in chronically instrumented goats. In control goats, MAP duration at 80% repolarization (MAPD80) shortened (P < 0.01) from 132+/-4 msec during slow pacing (400-msec interval) to 86+/-10 msec during fast pacing (180 msec). After cardioversion of sustained AF, the MAPD80 during slow pacing was as short as 67+/-5 msec (electrical remodeling). Increasing the pacing rate resulted in prolongation (P = 0.02) of the MAPD80 to 91+/-6 msec. Also, MAPD20 (20% repolarization) shortened (P = 0.05) from 32+/-4 msec (400 msec) to 14+/-7 msec (180 msec) in the control goats, whereas it prolonged (P = 0.03) from 20+/-3 msec (400 msec) to 33+/-5 msec (180 msec) in sustained AF. mRNA expression of the L-type Ca2+ channel alpha1c gene and Kv1.5 potassium channel gene, which underlie ICa and IKur, respectively, was reduced in sustained AF compared with sinus rhythm by 32% (P = 0.01) and 45% (P < 0.01), respectively. No significant changes were found in the mRNA levels of the rapid Na+ channel, the Na+/Ca2+ exchanger, or the Kv4.2/4.3 channels responsible for Ito. CONCLUSION: AF-induced electrical remodeling in the goat comprises shortening of MAPD and reversal of its physiologic rate adaptation. Changes in the time course of repolarization of the action potential are associated with changes in mRNA expression of the alpha subunit genes of the L-type Ca2+ channel and the Kv1.5 potassium channel.

The effect of the PKC inhibitor GF109203X on the release of Ca2+ from internal stores and Ca2+ entry in DDT1 MF-2 cells.

1. The effects of the specific protein kinase C (PKC) inhibitor, GF109203X, were measured on the cytoplasmic Ca2+ concentration ([Ca2+]i), and on histamine H1 receptor- and thapsigargin-mediated increases in [Ca2+]i in DDT1 MF-2 smooth muscle cells. 2. After pretreatment of cells with GF109203X (5 microM, 45 min), the histamine (100 microM)-induced initial rise in [Ca2+]i, representing Ca2+ mobilization from internal stores, was inhibited (by 59 +/- 7%). The slowly declining phase of the histamine induced Ca2+ response, reflecting Ca2+ entry, was enhanced (83 +/- 26%) in the presence of the PKC inhibitor. 3. The histamine induced release of Ca2+ from internal stores, measured after blocking Ca2+ entry with LaCl3 was inhibited by GF109203X in a concentration-dependent manner (IC50: 3.1 +/- 1.1 microM). 4. Histamine-induced formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) was not changed in the presence of GF109203X. 5. The PKC activating phorbol ester, phorbol 12-myristate 13-acetate (PMA, 1 microM), strongly reduced histamine-induced Ins(1,4,5)P3 formation (58 +/- 16%). This effect was reversed by GF109203X (5 microM). Furthermore, PMA diminished histamine evoked Ca2+ release (50 +/- 6%) and blocked Ca2+ entry completely. 6. The rise in [Ca2+]i caused by blocking endoplasmic reticulum Ca2(+)-ATPase with thapsigargin (1 microM), was strongly reduced (57 +/- 3%) after pretreatment of cells with GF109203X. Downregulation of PKC by long-term pretreatment of cells with PMA (1 microM, 48 h) did not abolish this effect of GF109203X (48 +/- 3% inhibition). 7. In permeabilized DDT, MF-2 cells preloaded with 45Ca2+ in the presence of GF109203X, the amount of 45Ca2+ released by Ins(1,4,5)P3 (10 microM) was markedly reduced (42 +/- 9%). GF109203X did not release Ca2+ itself and did not impair Ins(1,4,5)P3 receptor function. 8. Uptake of 45Ca2+ by intact cells, representing Ca2+ entry, was enhanced by GF109203X (65 +/- 11%), by histamine (24 +/- 6%) and also by thapsigargin (121 +/- 10%). The GF109203X- and the thapsigargin-induced uptake of 45Ca2+ were not additive. 9. These data suggest that GF109203X reduces the filling-state of intracellular Ins(1,4,5)P3 sensitive Ca2+ stores by inhibiting the Ca2+ uptake into these stores, thereby promoting store-dependent (capacitive) Ca2+ entry.

Neomycin inhibits histamine and thapsigargin mediated Ca2+ entry in DDT1 MF-2 cells independent of phospholipase C activation.

The histamine H1 receptor mediated increase in cytoplasmic Ca2+ ([Ca2+]i) was measured in the presence of the known phospholipase C (PLC) inhibitor, neomycin. Neomycin (1 mM) inhibited the histamine (100 microM) induced rise in [Ca2+]i to the same extent as observed after blocking Ca2+ entry with LaCl3. Likewise, the increase in [Ca2+]i after re-addition of CaCl2 (2 mM) to extracellular Ca2+ deprived and histamine pretreated cells was strongly reduced by neomycin. However, neomycin did not inhibit the histamine induced formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) or the release of Ca2+ from internal stores. These results show that neomycin blocks histamine induced Ca2+ entry independent of phospholipase C activation. Inhibition of intracellular store Ca(2+)-ATPase by thapsigargin (1 microM), elicited an increase in [Ca2+]i due to a leakage from the stores, subsequently followed by store-dependent Ca2+ entry. Thapsigargin induced Ca2+ entry was also completely blocked by neomycin. These results indicate that neomycin inhibits histamine and thapsigargin induced Ca2+ entry. This inhibition is most likely exerted at the level of plasma membrane Ca2+ channels.

The role of inositol 1,3,4,5-tetrakisphosphate in internal Ca2+ mobilization following histamine H1 receptor stimulation in DDT1 MF-2 cells.

Receptor-activated formation of inositol phosphates results in mobilization of intracellular stored Ca2+ in a variety of cells, including vas deferens derived DDT1 MF-2 cells. Stimulation of the histamine H1 receptor on these cells caused a pronounced formation of inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) with respect to that of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). In this study, the role of inositol phosphates, in particular Ins(1,3,4,5)P4 on the internal Ca(2+)-releasing process was investigated in permeabilized and histamine-stimulated intact DDT1 MF-2 cells. In permeabilized cells. Ins(1,4,5)P3 induced a concentration-dependent release of intracellular stored Ca2+. Addition of Ins(1,3,4,5)P4 did not cause Ca2+ mobilization, but its presence enhanced the amount of Ca2+ released by Ins(1,4,5)P3, thereby increasing the total Ca(2+)-releasing capacity. The effect of both inositol phosphates was inhibited by heparin, known to block Ins(1,4,5)P3-sensitive receptors. Thus, the additional amount of Ca2+ released by Ins(1,3,4,5)P4 is mediated, either via Ins(1,4,5)P3-sensitive Ca2+ channels, or via different heparin-sensitive Ca2+ channels activated by both Ins(1,4,5)P3 and Ins(1,3,4,5)P4. Histamine H1 receptor stimulation in intact cells induced a Ca(2+)-dependent K+ current, representing Ca2+ release from internal stores if receptor-activated Ca2+ entry from the extracellular space was prevented under Ca(2+)-free conditions or in the presence of La3+. This transmembrane current was abolished in the presence of intracellularly applied heparin. Depletion of Ins(1,4,5)P3-sensitive Ca2+ stores by internal application of Ins(1,4,5)P3 reduced the histamine evoked K+ current to some extent if the contribution of external Ca2+ was excluded.(ABSTRACT TRUNCATED AT 250 WORDS)

Arachidonic acid is functioning as a second messenger in activating the Ca2+ entry process on H1-histaminoceptor stimulation in DDT1 MF-2 cells.

This study was carried out to identify the cellular component activating the histamine-stimulated Ca2+ entry in vas-deferens-derived DDT1 MF-2 cells. H1-histaminoceptor stimulation resulted in a rise in intracellular Ca2+ concentration, caused by Ca2+ release from inositol phosphate-sensitive Ca2+ stores and Ca2+ entry from the extracellular space, accompanied by a transient Ca(2+)-activated outward K+ current. The histamine-evoked K+ current was still observed after preventing inositol phosphate-induced Ca2+ mobilization by intracellularly applied heparin. This current was activated by Ca2+ entry from the extracellular space, because it was abolished in the presence of the Ca(2+)-channel blocker La3+ or under Ca(2+)-free conditions. H1-histaminoceptor-activated Ca2+ entry was also observed in the presence of intracellularly applied Ins(1,4,5)P3 and Ins(1,3,4,5)P4, depleting their respective Ca2+ stores and pre-activating the inositol phosphate-regulated Ca2+ entry. Thus the ability of histamine to activate Ca2+ entry independently of Ca2+ mobilization and the formation of inositol phosphates suggests that another component is involved to initiate the Ca(2+)-entry process. It was observed that H1-histaminoceptor stimulation resulted in a pronounced release of arachidonic acid (AA) in DDT1 MF-2 cells. Exogenously applied AA induced a concentration-dependent increase in internal Ca2+ due to activation of Ca2+ entry from the extracellular space. Slow inactivation of the AA-sensitive Ca2+ channels is suggested by the slow decline in Ca2+ entry. In accord, the histamine-induced Ca2+ entry was not observed with AA-pre-activated Ca2+ channels. Inhibition of the lipoxygenase and cyclo-oxygenase pathway did not affect the AA-induced Ca2+ and the concomitant K+ current were decreased in the presence of AA and caused by Ca2+ mobilization from internal stores. Blocking this internal Ca2+ release by heparin, in the presence of AA, resulted in abolition of the histamine-induced Ca(2+)-regulated K+ current. These observations show that AA, released on H1-histaminoceptor stimulation in DDT1 MF-2 cells, is functioning as a second messenger to activate plasma-membrane Ca2+ channels promoting Ca2+ entry from the extracellular space.

Nucleotide receptors on DDT1 MF-2 vas deferens cells.

On exposure to triphosphatic nucleotides vas deferens DDT1 MF-2 smooth muscle cells responded with an outward K+ current as measured with the whole-cell patch clamp configuration. The rank order of potency was: ATP greater than UTP greater than TTP greater than CTP = GTP. The responses evoked by these agonists were blocked by suramin. Adenosine, ADP, alpha, beta-methylene-ATP and 2-methylthio-ATP did not affect the transmembrane current. The responses evoked by the nucleotides in DDT1 MF-2 cells are supposed to be mediated via 'nucleotide' receptors.