Molecular characterization of glucose-6-phosphate dehydrogenase deficiency in Pakistani population.

INTRODUCTION: Glucose-6-phosphate dehydrogenase (G6PD; E.C. 1.1.1.49) deficiency is the commonest inborn error of metabolism with more than 140 genetic variants. The incidence of G6PD deficiency is 2-9% in Pakistan, but G6PD variants were never studied comprehensively. We therefore designed this study to describe the frequency of G6PD variants and their associated enzyme activities in Pakistan. METHODS: Patients diagnosed with G6PD deficiency were enrolled. RFLP-PCR was utilized to identify common mutations previously reported from Asian countries. Where mutational analysis failed, amplification of 9-12 exons with subsequent gene sequencing was performed. G6PD enzyme activity was assessed through the quantitative enzyme assay. RESULTS: Two hundred and seventy-six G6PD-deficient subjects (237 male and 39 women) were investigated. G6PD Mediterranean (563C-T) was the most common genetic variant (n=216 or 78%). G6PD Chatham (1003A-G) and G6PD Orissa (131C-G) were observed in 14 (5%) and two (0.7%) subjects respectively. A novel mutation 973 G-A with a predicated amino acid change of asp325asn was identified in exon 9. This was named G6PD Karachi after the place of origin of proband. Polymorphism in position 1311C/T was uniformly observed with all variants. Forty-three or 17% of DNA samples remained uncharacterized. Very low levels of G6PD enzyme activity was observed with 563C-T mutation. CONCLUSION: We concluded that 563C-T was the commonest G6PD variant, while 1003A-G and 131C-G were less-frequent genetic variants of G6PD in Pakistani population. A novel genetic variant 973G-A was also identified. Very low levels of G6PD enzyme activity was seen with G6PD 563C-T. Mutational analysis failed in a significant proportion of samples warranting further work.

Molecular analysis of human leukocyte antigen class I and class II allele frequencies and haplotype distribution in Pakistani population.

AIM: Distribution of HLA class I and II alleles and haplotype was studied in Pakistani population and compared with the data reported for Caucasoid, Africans, Orientals and Arab populations. MATERIALS AND METHODS: HLA class I and II polymorphisms in 1000 unrelated Pakistani individuals was studied using sequence-specific primers and polymerase chain reaction and assay. RESULTS: The most frequent class I alleles observed were A*02, B*35 and CW*07, with frequencies of 19.2, 13.7 and 20%, respectively. Fifteen distinct HLA-DRB1 alleles and eight HLA-DQB1 alleles were recognized. The most frequently observed DRB1 alleles which represented more than 60% of the subjects were DRB1 *03, *07, *11 and *15. The rare DRB1 alleles detected in this study were HLADRB1 *08 and *09, having frequencies of 0.9 and 1.7%, respectively. In addition, at DRB1-DQB1 loci there were 179 different haplotypes and 285 unique genotypes and the most common haplotype was DRB1*15-DQB1*06 which represented 17% of the total DRB1-DQB1 haplotypes. In our population, haplotype A*33-B*58-Cw*03 comprised 2.8% of the total class I haplotypes observed. This haplotype was seen only in the oriental populations and has not been reported in the African or European Caucasoid. CONCLUSION: Our study showed a close similarity of HLA class I and II alleles with that of European Caucasoid and Orientals. In Pakistani population, two rare loci and three haplotypes were identified, whereas haplotypes characteristic of Caucasians, Africans and Orientals were also found, suggesting an admixture of different races due to migration to and from this region.

Increased expression of HLA DR2 in acquired aplastic anemia and its impact on response to immunosuppressive therapy.

OBJECTIVE: To study the frequency of HLA DR2 status of patients with aplastic anemia and their response to immunosuppressive therapy at a tertiary care hospital. METHODS: Thirty eight consecutive patients of acquired aplastic anemia were evaluated with respect to demographic features, severity of HLA DR2 status and response outcome to immunosuppressive therapy. RESULTS: The mean age of the patients was 24.6 years + 16.4 with a male to female ratio of 2.8:1. Positivity of HLA DR2 was markedly high in acquired aplastic anemia patients. Twenty four (65%) out of 38 patients as compared to 45 (15%) of 300 healthy controls (p<0.0001) were positive for HLA DR2. Response to immunosuppressive therapy, which included antilymphocyte globulin, cyclosporin and methylprednisolone, was available in sixteen HLA DR2 positive patients and was found satisfactory in 12/16 (75%) patients. CONCLUSION: HLA DR2 was significantly higher in patients with acquired aplastic anemia and favourable response to immunosuppressive therapy was also associated with HLA DR2 positivity.

Interleukin (IL) 1beta and IL-10 gene polymorphism in chronic hepatitis C patients with normal or elevated alanine aminotransferase levels.

OBJECTIVE: To evaluate the frequency of cytokine gene single nucleotide polymorphisms (SNP) of IL-1beta and IL-10 at promoter sites -115 and -1082 respectively and their association with hepatocellular injury as suggested by alanine aminotransferase (ALT) level. METHODS: Fifty six patients (31 males, 25 females) positive for HCV antibody were studied. Their mean age was 37.5 +/- 12.0 years (range 16-62). Fifty one were HCV RNA positive; 14 had normal and 37 elevated ALT levels. Type 3 was the predominant genotype. For cytokine polymorphism DNA was extracted from the whole blood. After amplification with polymerase chain reaction, biallelic polymorphisms of IL-1beta at -115 and IL-10 at -1082 promotor site were determined using sequence specific oligonucleotide primers. RESULTS: IL-1beta-15 C allele was present in 49 (87.5%) and -115 T in 30 (53.6%) patients. Genotype CC was present in 26 (46%), TC 23 in (42%), and TT in 07 (12%). IL-10 -1082 A allele was seen in 50 (89.3%) and -1082 G in 35 (62.5%). Genotype AG in 29 (52%), AA in 21(37%), and GG in 06 (11%). Though greater number of HCV RNA positive patients with normal ALT had IL-10 -1082 AG genotype (10 out of 14), no statistically significant difference could be found in the genotype profile of the above promoter sites of IL-1beta and IL-10 in patients with normal or elevated ALT. CONCLUSION: IL-1beta-115 C and IL-10 -1082A are the more frequent alleles in our patients. Genotypes L-1beta -115 CC and TC, and IL-10 -1082AG and AA are common while -115 TT and -1082 GG are less common. Any effect of IL-1beta and IL-10 gene polymorphism on the degree of hepatocellular injury may not be apparent by the ALT levels.

Characterization of pathogenic mutations in 21-hydroxylase gene of Pakistani patients with congenital adrenal hyperplasia and their family members--a preliminary report.

OBJECTIVE: To characterize specific mutations within the 21-hydroxylase gene (CYP21-B) using ARMS-PCR assay in patients with congenital adrenal hyperplasia (CAH) and to compare it with that reported in other populations. SUBJECTS AND METHODS: Five families, having an index case with CAH diagnosed on the basis of clinical and biochemical findings volunteered to give blood samples for analysis. A strategy, based on ARMS-PCR (Amplified Refractory Mutation System) was employed for the detection of mutations in 21-hydroxylase gene. The products of ARMS-PCR were resolved on agarose gels and the PCR products were visualized over ultra violet illumination. RESULTS: Twenty-six specimens were analyzed for common point mutations in the 21-hydroxlase genes at the nucleotide positions 659, 1004 and 1688. Seven samples belonged to index cases with CAH. Of these 7, the assigned sex was male in 5 and female in 2 cases. However, genotypic sex was 3 males and 4 females. The mean age was 8 months in 5 cases while the median 17-OH Progesterone levels was 273.2 ng/ml. Vomiting, precocious puberty and ambiguous genitalia were the presenting features in 2, 1 and 4 cases respectively. Analysis for mutation at 659, 100 and 1688 was performed on 7 index cases and the family members of 5 index cases. The mutation analysis for the family members of index case 6 and 7 was not performed due to non-availability of their blood specimens. Index case No. 1, 4 and 7 showed homozygosity for splice mutations at nucleotide position 659, intron 2 with a sequence change of A to G, while the index case No. 2 and 6 showed heterozygosity for the same mutation. No mutation was found at 659, 1004 or 1688 in index case No. 3 and 4 at the analyzed nucleotide position. Nineteen family members of Case Nos. 1-5 were also analyzed for the same mutations. (Family No. 1, 2, 3, 4 and 5 included 3, 2, 7, 4 and 5 members respectively). These included 8 males and 11 females. All were asymptomatic. Both the parents of index case 1 and 4 were heterozygous at 659 while the father of index case No. 2 was heterozygous at 659 and mother was normal. CONCLUSION: Our results demonstrated the A to G transition at nucleotide 659 causing aberrant splicing, reported for some other populations as the most commonly identified point mutations. All cases were appropriately assigned to paternal or maternal chromosomes.

Prevalence and risk factors for intrafamilial transmission of hepatitis C virus in Karachi, Pakistan.

Household contacts of hepatitis C virus (HCV)-positive patients are considered at increased risk of HCV infection. This cross-sectional study during April through June 1999 assessed the prevalence and risk behaviours associated with HCV seropositivity among the household contacts of HCV seropositive thalassaemic children in Karachi, Pakistan. Among the 341 household contacts of 86 thalassaemic HCV seropositive children who were tested, 70 (20.5%) were positive for anti-HCV antibodies. The stratified analysis showed that HCV seroprevalence among the contacts did not differ significantly by the gender of the index patient and the type of relationship of contact with the index patient. However, HCV seroprevalences among the fathers and mothers of male index patients was substantially higher compared to those of female index patients. HCV RNA was recovered and genotyped from nine index patients and corresponding nine HCV-seropositive household contacts. HCV genotype 3a and 3b were found in 89% (8/9) and 11% (1/9) of the pairs, respectively. The final multivariable conditional logistic regression model revealed that after adjusting for the effect of ethnicity and past hospital admission history, the HCV-seropositive household contacts were more likely than HCV seronegative household contacts to have been bitten by the carrier [adjusted matched odds ratio (mOR)=2.6, 95% CI 1.3-5.2] or have shared a toothbrush with the carrier (adjusted mOR=8.2; 95% CI 1.56-43.5). Control efforts should focus on the risk behaviours.

Genetic diversity of beta-thalassemia mutations in Pakistani population.

BACKGROUND: beta-thalassemia is one of the most common inherited single gene disorder in Pakistan. It is characterized by reduced or absent beta-globin gene expression resulting in abnormal maturation and survival of red blood cells. Due to high prevalence of this disease in the local population, it has become important for the health care providers to encourage people to utilize laboratory facilities for carrier and prenatal genetic testing. OBJECTIVE: To study the frequency of beta-thalassemia mutations in Pakistani population. SETTING: A tertiary care teaching hospital. METHODS: Blood samples of 72 couples and chorionic villus (CV) biopsy specimen collected at the Aga Khan University Hospital, Karachi were tested by Amplified Refractory Mutation Systems (ARMS) for the 12 most common mutations in the beta-globin gene. RESULTS: Out of 72 chorionic villus biopsy specimen analyzed, 17 (23%) had mutations in both alleles of the beta-globin gene. Homozygosity was identified in 6 CV samples, whereas 11 CV specimens were diagnosed as double heterozygous. Almost 60% of the CV biopsies showed mutations in one allele and were diagnosed as carriers. IVSI-5 (G-C) was the most common mutation identified in this study. It was found in 53% of the subjects and was represented equally in all the ethnic groups except Pathans. Several regional and ethnic differences were observed in the distribution of common mutations, for example in Pathan families Fr 8-9 (+G) mutation was most prevalent. In addition, variation in the distribution of mutations was also observed between the Northern and the Southern regions. CONCLUSION: This study indicates that in Pakistan, the five most common mutations are IVS1-5 (G-C), IVS1-1 (G-T), Fr 41-42 (-TTCT) Fr 8-9 (+G) and deletion 619 bp. An important factor contributing to high incidence of thalassemia is the unawareness among people about the available diagnostic facilities for the prenatal diagnosis in Pakistan. Strict implementation of collective measures including carrier identification, genetic counseling and prenatal diagnosis are required for preventing beta-thalassemia in Pakistan.

Association of Epstein Barr Virus (EBV) with nasopharyngeal carcinoma (NPC).

AIM: To observe the frequency of nasopharyngeal carcinoma (NPC) and its association with Epstein Barr Virus (EBV) infection. SETTING: This study included consecutive cases of nasopharyngeal carcinoma, which were diagnosed in the Department of Pathology at the Aga Khan University Hospital, Karachi in the period of two years (1996-97). METHODS: These tumors were initially evaluated on H&E stained sections. The tumors showing evidence of keratinization were excluded from the study. The Epstein Barr Virus was detected with the help of Polymerase chain reaction in formalin fixed, paraffin embedded tissue sections. RESULTS: During the study period, seventeen cases of nasopharyngeal carcinoma were diagnosed which comprised 0.3% of all malignant tumors. The age ranged from 5 years to 70 years with male to female ratio of 2.4:1. The NPC was more prevalent in adults (71%) as compared to children (29%) under 15 years. Six cases (35%) exhibited positive signal for Epstein Barr Virus. CONCLUSION: Nasopharyngeal carcinoma is an infrequent tumor. The prevalence of Epstein Barr virus infection in nasopharyngeal carcinoma is quite low as compared to other regions of the world.

Prevalence of hepatitis G virus in Pakistani children with transfusion dependent beta- thalassemia major.

We ought to obtain data on the prevalence of the newly discovered tranfusion transmittable hepatitis G virus in polytransfused b- thalassemia major children. Each individual had received multiple blood transfusions, from 12 to 36 per year. No documentation of prior hepatic infection was available. Serum samples were collected prospectively from the randomly selected subjects and were analyzed for HGV RNA by polymerase chain reaction using primer specific for two different regions of the HGV genome. Among the 100 individuals examined 21 were positive for HGV RNA. Four patients had evidence of dual infection, both HGV RNA and HCV RNA were isolated from their sera. While in one sample presence of both HGV RNA and HBV DNA was established. Only one child was positive for hepatitis E antibodies. The sera of 10 children were reactive for hepatitis B surface antigen whereas 35 individuals were positive for hepatitis C virus antibody. The ALT levels were variable in HGV infected children. Four out of 16 (25%) showed peak ALT levels of 218 IU/I, 8/16 (50%) children demonstrated slightly elevated ALT levels whereas 25% individuals showed normal ALT levels. Alkaline Phosphatase levels were elevated in 90% of the children and 20% patients of this series also had higher GGT levels. The observed AP levels were not statistically different among HGV, HGV/HCV or HGV/HBV groups. Even though the ALT levels were deranged in the children with HGV alone but none of the children had demonstrated symptoms of liver disease, their direct and total bilirubin levels were normal and no complain of jaundice was recorded. In conclusion, our findings suggested that like other blood borne hepatic viruses, HGV is also prevalent in the high risk group of multiple transfused patients in Pakistan but our results support the absence of any causal relationship between HGV and hepatitis.

Detection of Mycobacterium tuberculosis in paraffin embedded intestinal tissue specimens by polymerase chain reaction: characterization of IS6110 element negative strains.

Tuberculosis is still one of the most widespread infection known to mankind. Although lung is the predominant site of disease, a sizeable population in Pakistan gets intestinal disease. Clinical presentation, radiologic and endoscopic examination provide clues to the diagnosis. However, a definitive diagnosis requires biopsy material with granulomas and/or caseation complemented by acid fast staining and culture. There are many occasions when biopsy material is scanty and even in some intestinal resection cases histologic evaluation fails to confirm or rule out tuberculosis. Therefore, an investigation was conducted to assess the efficacy of PCR in the detection of mycobacterial DNA in paraffin embedded intestinal tissue. In this study 12 histologically confirmed cases of intestinal tuberculosis and 2 cases with non specific inflammation but clinically suspected for abdominal tuberculosis were selected. One case of rectal polyp was included to serve as a negative control. M. tuberculosis DNA was amplified in 8 out of 12 histologically confirmed cases and in 2 cases diagnosed with non specific inflammation. Amplified products were obtained in 6 out of 10 PCR positive specimens with IS6110 region specific primers while 4 samples were negative, suggesting the absence of insertion sequence 6110 in these strains. However, amplification was obtained in these negative specimens with a second primer pair confirming them as M. tuberculosis complex species. On the basis of this study we conclude that; (1) Processed and paraffin embedded tissue material is suitable for PCR analysis, (2) PCR assay can be used to complement the diagnosis of intestinal tuberculosis especially in situations where a definite conclusion can not be drawn by conventional methods, (3) M. tuberculosis species lacking insertion sequence 6110 element are present in our population. Therefore, several primer pair sets should be included when applying PCR for the detection of mycobacterial DNA.

Induction of cyclooxygenase II in human synovial microvessel endothelial cells by interleukin-1. Inhibition by glucocorticoids.

OBJECTIVE: To characterize the effects of interleukin-1 alpha (IL-1) on prostanoid biosynthesis by human rheumatoid synovium microvessel endothelium (HSE). METHODS: HSE cells were treated with cytokines, metabolic inhibitors, and steroids under various conditions, and prostaglandin biosynthesis was determined by radioimmunoassay. Newly synthesized cyclooxygenase (COX) was quantitated by immunoprecipitation of metabolically labeled HSE cell lysates. The effects of IL-1 on levels of messenger RNA (mRNA) for COX II were also determined. RESULTS: IL-1 induced an increase in COX activity (as assessed by prostaglandin E2 release) that was dose- and time-dependent and was blocked by cycloheximide, actinomycin D, and dexamethasone. IL-1 induced a selective increase in COX II mRNA and biosynthesis of COX II protein that was blocked by dexamethasone. CONCLUSION: IL-1 treatment of HSE cells induces COX II, as demonstrated by both Northern blotting and immunoprecipitation. The induction of COX II expression provides, at least in part, a mechanism for the pronounced increase in prostanoid synthesis observed in HSE cells following incubation with IL-1. The selective up-regulation of HSE COX II by inflammatory cytokines such as IL-1 suggests that development of specific pharmaceutical inhibitors for this novel isozyme may provide significant new therapeutic advantages in the treatment of RA.

Acidic fibroblast factor induction of cyclooxygenase II in rabbit cardiac muscle microvessel endothelial cells: mediation by protein kinase C.

OBJECTIVE: To determine if the novel cyclooxygenase, COX II, was induced by acidic fibroblast growth factor (aFGF) in rabbit cardiac muscle microvessel (RCME) endothelial cells and to determine the role of protein kinase C (PKC) activation in the mediation of the aFGF induction of COX activity. METHODS: Cultured RCME cells were treated with aFGF (200 ng/ml). Induction of COX II activity was assessed by determination of COX activity (PGE2 production), by immunoprecipitation of metabolically labeled COX II, and by Northern analyses. The role of PKC was assessed using phorbol myristate acetate and PKC inhibitors and by determination of PKC activity in cytosol and membrane fractions of RCME cells treated with aFGF and phorbol myristate acetate. RESULTS: aFGF selectively induced COX II protein and mRNA. Protein kinase C activation was implicated in the transduction of the effects of aFGF for the following reasons: (1) phorbol myristate acetate (PMA), a direct activator of protein kinase C, was a potent inducer of COX II mRNA, COX activity and synthesis of COX II protein. (2) H-7, an inhibitor of PKC, but not the inactive control, HA-1004, blocked aFGF induction of COX II mRNA, COX II protein synthesis, and COX activity. Two additional inhibitors of PKC, calphostin C and staurosporine, also inhibited aFGF induction of COX activity. (3) Downregulation of PKC by overnight incubation with 1 microM PMA blocked subsequent induction of COX II protein synthesis by aFGF. (4) aFGF treatment of RCME cells resulted in the translocation of PKC activity from the cytosol to the membrane fraction. However, aFGF, at concentrations that elicited COX II, neither induced Ca2+ mobilization from intracellular stores nor increased the accumulation of inositol phosphates. CONCLUSION: aFGF induces COX II in RCME and this response in mediated, at least in part, by protein kinase C activation. However, aFGF mediated activation of PKC activation must stimulate this kinase through a pathway of signal transduction distinct from inositol phospholipid accumulation or elevation of intracellular Ca2+.

Cellular signaling responses mediated by a novel nucleotide receptor in rabbit microvessel endothelium.

The adenine nucleotide, ATP, elicits an elevation in intracellular ionized calcium concentration ([Ca2+]i) and phospholipase C-mediated phosphatidylinositol hydrolysis and stimulates the synthesis of the prostaglandins E2 and I2 in cultured endothelial cells derived from rabbit cardiac muscle. Use of various ATP analogues indicated that these events did not fit the classical definition of P1 or P2 purinergic receptors and, furthermore, indicated that the receptor(s) mediating these activities was not specific for purines. The rank order of agonist potency on prostaglandin release, elevations in [Ca2+]i, and inositol phosphate response was UTP > or = ATP > ADP > ADP[beta]S = 2-methylthio ATP > adenosine, suggesting that these three cellular responses are coupled to the same or similar receptors. However, the sensitivity of these three cellular responses to added nucleotides was somewhat different. The half-maximum effective concentration (EC50) for ATP stimulation of prostaglandin release was 100 microM, for inositol phosphate turnover it was 25 microM, and for elevations in [Ca2+]i it was < 1 microM. Similar discrepancies in EC50 UTP values for these three cellular responses were also noted. These observations indicate that purine and pyrimidine nucleotides elicit at least three cellular responses in rabbit cardiac muscle microvessel endothelial cells, all demonstrating similar rank orders of potency. However, the differences in EC50 suggest that if these responses are mediated by a single receptor type, it exhibits divergent coupling to various cellular signaling pathways.

Fibroblast growth factor upregulates PGG/H synthase in rabbit microvascular endothelial cells by a glucocorticoid independent mechanism.

The present study was undertaken to determine the effects of acidic fibroblast growth factor (aFGF) on eicosanoid synthesis in microvessel endothelial cells derived from rabbit left ventricular muscle (RCME cells). We observed that aFGF increased AA conversion to PGE2 in a time- and dose-dependent manner, and the stimulatory effect was abolished by actinomycin D and cycloheximide. Acidic FGF increased the recovery of PGG/H synthase activity following aspirin treatment, suggesting an action on de novo PGG/H synthase synthesis. Acidic FGF increased the incorporation of [35S] methionine into a 70 kD immunoreactive PGG/H synthase band. PGG/H synthase synthesis following aspirin treatment was also increased by transforming growth factor beta, while epidermal growth factor basic FGF and platelet derived growth factor were without effect. In addition, the actions of aFGF on de novo PGG/H synthase were compared in several endothelial preparations. Acidic FGF treatment of aspirin treated endothelial cells from rabbit lung microvessels and small pulmonary artery and from human lung microvessels all showed an increase in PGG/H synthase recovery. In contrast, similar treatment of human umbilical vein endothelial cells was without effect. Pretreatment of RCME cells with dexamethasone (1 microM) did not alter the aFGF induction of PGG/H synthase activity. We conclude that aFGF stimulates PGE2 production by a mechanism that includes the de novo synthesis of PGG/H synthase. This mechanism appears to be distinct from previously described glucocorticoid sensitive translational controls of PG synthase synthesis by epidermal growth factor in smooth muscle and mesangial cells.

Dexamethasone effects on microvascular endothelial cell lipid composition.

Previous studies have shown that treatment of cultured rabbit coronary microvessel endothelial (RCME) cells with dexamethasone results in a dose-, time-, and glucocorticoid-dependent inhibition of prostaglandin release. In the present study, the effects of dexamethasone on RCME membrane lipid composition and release of arachidonic acid were examined. This study demonstrated that dexamethasone treatment did not significantly alter the relative distribution of membrane phospholipids but did result in changes of fatty acid composition. There was an increase in saturated and monounsaturated fatty acids and a decrease in polyunsaturated fatty acids. Dexamethasone treatment did not reduce A23187-stimulated arachidonic acid release, despite inhibiting prostaglandin release by 50%. Studies with radiolabeled arachidonic acid suggest that dexamethasone may exert some actions on membrane remodeling, an effect that will require further investigation. Our data strongly suggest that the inhibitory actions of glucocorticoids on prostaglandin release in cultured RCME cells are not the result of a generalized inhibition of arachidonic acid release, and alternate mechanisms must therefore be considered.

Agonist-specific role for Na+-H+ antiport in prostaglandin release from microvessel endothelium.

Rabbit coronary microvascular endothelial (RCME) cells synthesize prostaglandin (PG) I2 and PGE2 in response to stimulation with human thrombin, ATP, and the Ca2+ ionophore, A23187. Replacement of extracellular Na+ with choline or N-methylglucamine reduced thrombin-stimulated PG secretion but did not significantly affect either ATP- or A23187-stimulated PG secretion. Pretreatment of RCME cells with Na+ channel or Na+ -Ca2+ exchange blockers did not alter PG release in response to any of these three agonists. Pretreatment of RCME cells with the specific Na+ -H+ antiport blockers 5-(N-ethyl-N-isopropyl)-amiloride (EIPA, 10 microM) and 5-(N,N-hexamethylene)-amiloride (HMA, 0.1 microM) significantly reduced thrombin but not A23187- or ATP-stimulated PG secretion. Studies with the intracellular pH indicator dye 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein demonstrated thrombin activation of Na+ -H+ antiport, an effect blocked by either HMA or EIPA. Since manipulations known to inhibit Na+ -H+ exchange (EIPA, HMA, replacement of Na+ with choline or N-methylglucamine) reduced thrombin-stimulated RCME cell PG release, we conclude that activation of Na+ -H+ exchange is involved in the coupling of thrombin interaction with RCME cells to subsequent phospholipase activation and PG release.