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Biomedical subjects

T Iwamoto

Publications and source records attributed to T Iwamoto.

At least 127 records · Page 7Linked to original sources

Unique topology of the internal repeats in the cardiac Na+/Ca2+ exchanger.

Hydropathy analysis predicts 11 transmembrane helices in the cardiac Na+/Ca2+ exchanger. Using cysteine susceptibility analysis and epitope tagging, we here studied the membrane topology of the exchanger, in particular of the highly conserved internal alpha-1 and alpha-2 repeats. Unexpectedly, we found that the connecting loop in the alpha-1 repeat forms a re-entrant membrane loop with both ends facing the extracellular side and one residue (Asn-125) being accessible from the inside and that the region containing the alpha-2 repeat is mostly accessible from the cytoplasm. Together with other data, we propose that the exchanger may consist of nine transmembrane helices.

Animals↗

Spermatogenic ability is different among males in different Y chromosome lineage.

It is a controversial question whether sperm concentrations in humans are changing. Several researchers have reported on environmental factors affecting sperm quality, but the influence of genetic factors is still not fully understood. In this study, we examined the relationship between Y chromosome haplotypes and sperm concentration in fertile males. In addition, we determined the haplotypes of azoospermic patients. The results show that the mean sperm concentration correlates with Y chromosome type. Moreover, the occurrence of azoospermia is related to one particular Y chromosome lineage. Thus, males with a certain haplotype are at a disadvantage for fathering children. The difference of spermatogenic ability among men is important not only in pursuing male competition as in the past but also as relates to the future of modern human males.

Fertility↗

Thoughts and feelings of in-patients with advanced terminal cancer: implications for terminal care improvement.

The purpose of this study was to clarify how terminally ill patients think and feel while hospitalized, thereby providing some insight into improving the quality of terminal care. The subjects of the study were eight terminally ill patients with cancer. The researchers obtained the patients' consent and their doctors' permission prior to conducting the interviews. Individual, semi-structured interviews were conducted. The patients' thoughts and feelings were extracted and then categorized according to particular themes. As a result, we came up with four major categories: maintaining pain control, living an ordinary life, hoping for improvement of physical condition, and having family nearby. The desire to live life to its fullest given their situation, as well as to maintain a hopeful attitude about their future proved very important. The presence of family also played a crucial role. These factors must be taken into consideration in achieving better care for terminally ill patients.

Aged↗

Angiotensin II inhibits interleukin-1 beta-induced nitric oxide production in cultured rat mesangial cells.

BACKGROUND: Macrophage-type nitric oxide synthase (NOS-II) is expressed in glomerular mesangial cells in response to inflammatory cytokines. Nitric oxide (NO) has antithrombotic and cytostatic activities in glomerular diseases. Recent studies have suggested that several vasoactive substances and growth factors modulate NO production in a tissue-specific manner. The aim of this study was to examine whether angiotensin II and transforming growth factor-beta (TGF-beta) modulate cytokine-stimulated NO production and NOS-II gene expression in rat glomerular mesangial cells. METHODS: Cultured rat mesangial cells were incubated with interleukin-1 beta (IL-1 beta) for 24 hours. The effects of angiotensin II and TGF-beta on stimulated nitrite accumulation and NOS-II mRNA levels were determined. RESULTS: Angiotensin II and TGF-beta significantly decreased IL-1 beta-stimulated nitrite accumulation. The angiotensin type 1 receptor antagonist CV11974 prevented angiotensin II-mediated inhibition of NO production. TGF-beta-neutralizing antibody reversed the effect of TGF-beta without affecting angiotensin II-mediated inhibition of NO production. TGF-beta markedly decreased steady-state levels of NOS-II mRNA and the half-life of the message, whereas angiotensin II did not alter these parameters. CONCLUSIONS: These results suggest that in mesangial cells, angiotensin II and TGF-beta participate in the inhibitory regulation of cytokine-induced NO production. TGF-beta inhibits NO production by decreasing NOS-II mRNA levels, whereas angiotensin II may regulate NO production at the levels after NOS-II gene expression. An autocrine action of TGF-beta induced by angiotensin II is unlikely to contribute to angiotensin II-mediated inhibition of NO production.

Angiotensin II↗

Sevoflurane requirements for tracheal intubation with and without fentanyl.

We studied 80 healthy ASA 1 patients (aged 20-52 yr) to determine if fentanyl affects sevoflurane requirements for achieving 50% probability of no movement in response to laryngoscopy and tracheal intubation (MAC-TI). Patients were allocated randomly to one of four fentanyl dose groups (0, 1, 2 and 4 micrograms kg-1). Patients in each group received sevoflurane at a pre-selected end-tidal concentration according to an 'up-down' technique. After steady state sevoflurane concentration had been maintained for at least 10 min, fentanyl was administered i.v. Tracheal intubation was performed 4 min after administration of fentanyl, and patients were assessed as moving or not moving. Heart rate (HR) and mean arterial pressure (MAP) were recorded before induction of anaesthesia, just before administration of fentanyl, just before laryngoscopy for intubation, and after intubation. The MAC-TI of sevoflurane was 3.55% (95% confidence intervals 3.32-3.78%), and this was reduced markedly to 2.07%, 1.45% and 1.37% by addition of fentanyl 1, 2 and 4 micrograms kg-1, with no significant difference in the reduction between 2 and 4 micrograms kg-1, showing a ceiling effect. Fentanyl attenuated haemodynamic responses (HR and MAP) to tracheal intubation in a dose-dependent manner, even with decreasing concomitant sevoflurane concentration. Fentanyl 4 micrograms kg-1 suppressed the changes in HR and MAP more effectively than fentanyl 1 or 2 micrograms kg-1 at sevoflurane concentrations close to MAC-TI.

Adult↗

The effect of fentanyl on sevoflurane requirements for somatic and sympathetic responses to surgical incision.

BACKGROUND: Fentanyl produces a reduction in the minimum alveolar concentration (MAC) of isoflurane and desflurane needed to blockade adrenergic response (BAR) to surgical incision in 50% of patients (MAC-BAR). MAC-BAR of sevoflurane and the reduction in MAC-BAR of sevoflurane by fentanyl have not been described previously. The purpose of this study was to determine the MAC and MAC-BAR reduction of sevoflurane by fentanyl with and without nitrous oxide (N2O). METHODS: Two hundred twenty-six patients were randomly assigned to one of two groups: a sevoflurane group and a sevoflurane/N2O group. Patients in each group were randomly assigned to one of five different fentanyl concentration subgroups. Patients were anesthetized with sevoflurane and fentanyl in the sevoflurane group and with sevoflurane, fentanyl, and N2O (66 vol%) in the sevoflurane/N2O group. Somatic and sympathetic responses to surgical incision were observed for MAC and MAC-BAR assessment at predetermined concentrations of sevoflurane. RESULTS: Fentanyl produced an initial steep reduction in the MAC and MAC-BAR of sevoflurane, with 3 ng/ml resulting in a 61% reduction in MAC and an 83% reduction in MAC-BAR. A ceiling effect was observed for MAC and MAC-BAR, with 6 ng/ml fentanyl providing only an additional 13% and 9% reduction in MAC and MAC-BAR, respectively. In the presence of 66 vol% N2O, MAC and MAC-BAR of sevoflurane were reduced with increasing concentrations of fentanyL A ceiling effect was not observed for reduction in MAC and MAC-BAR in the presence of N2O. CONCLUSIONS: MAC and MAC-BAR decreased similarly with increasing concentrations of fentanyl in plasma, showing an initial steep reduction followed by a ceiling effect. In the presence of N2O, MAC and MAC-BAR decreased similarly but did not exhibit a ceiling effect.

Adult↗

Electrophysiologic and blood-flow responses in the endocardium and epicardium to disopyramide and MS-551 during myocardial ischemia in the dog.

The aim of this study was to determine whether a quantitative relation exists between changes in regional myocardial blood flow (RMBF) and those in electrophysiologic determinants recorded via left ventricular endocardial and epicardial bipolar electrograms after administration of disopyramide (DP) and a class III antiarrhythmic drug, MS-551 (MS), during myocardial ischemia in the dog. Dogs were given DP (1 mg/kg, i.v., n = 14), MS (1 mg/kg, i.v., and 0.1 mg/kg/min, d.i.v., n = 13), or saline (n = 12). The effective refractory period (ERP) was determined by an S1-S2 extrastimulus method, and RMBF by a nonradioactive microsphere technique. The duration of regional electrograms (DRE) was measured as an indicator of conduction time in the myocardium. DP blunted ischemia-induced shortening of ERPs and lengthened DREs at the endocardial and epicardial sites, with a greater effect seen epicardially (p < 0.01 each). DP reduced RMBF, especially at the endocardial surfaces of the ischemic zone (p < 0.05). MS prolonged ERPs at the endocardial and epicardial sites in the ischemic and normal zones (p < 0.05-0.01), but there were no significant differences between the two sites. MS prolonged DREs (p < 0.05), but the magnitude of the prolongation of the DREs was similar to the values in the control group. MS had no effects on RMBF. DP treatment prolonged DREs at both sites in the ischemic zone more markedly than MS or saline treatment (p < 0.01 each). DP reduced RMBF at the endocardial site of the ischemic zone more markedly than MS or saline (p < 0.05 in each). Accordingly, MS prolonged ERPs, but did not increase disparities between endocardial and epicardial sites in the ischemic myocardium, whereas DP had a greater ERP-prolonging effect at the epicardial site than at the endocardial site. DP reduced endocardial RMBF more markedly than epicardial RMBF. These observations suggest that differences in ERPs between endocardial and epicardial ischemic myocardium caused by DP treatment are not due to the difference in RMBF reduction between the two tissue layers, and that DP and MS do not affect the same population of ion channel(s) when ERPs are prolonged.

Animals↗

Regional differences in diffusion abnormality in cerebral white matter lesions in patients with vascular dementia of the Binswanger type and Alzheimer's disease.

We investigated changes in water diffusion in the cerebral white matter of 14 patients with vascular dementia of the Binswanger type (VDBT) and ten patients with Alzheimer's disease (AD) with periventricular hyperintensity (PVH) lesions using diffusion-weighted magnetic resonance imaging (MRI) and studied the pathophysiological differences between white matter lesions found in these two conditions. Apparent diffusion coefficients (ADCs) in the anterior and posterior white matter and the genu and splenium of the corpus callosum were significantly higher in both groups of patients than in the 12 age-matched controls, and ADC values in VDBT and AD groups were almost the same. ADC ratios, defined as diffusion restricted perpendicular to the direction of nerve fibers, were also significantly higher in the patients than in the control subjects. However, there were regional differences in ADC ratios in the two conditions, with ratios in VDBT being higher in the anterior portions of the white matter but ratios in AD were higher in the posterior portions. The diffusion-weighted MRI technique may be useful in the differential diagnosis of VDBT and AD with white matter lesions.

Aged↗

Splenic contraction-induced reversible increase in hemoglobin concentration in intermittent hypoxia.

The effect of intermittent hypoxia (IHx) on blood hemoglobin concentration ([Hb]) and the underlying mechanisms were studied in rats exposed to 10% O2, 1 h/day, for up to 5 wk. IHx protocols with longer daily hypoxic exposure show persistent polycythemia; however, it is unknown whether [Hb] increases transiently during hypoxia in protocols without polycythemia. Hypoxia produced a reversible [Hb] increase after 4 days of IHx but not in normoxic controls (NxC) or after shorter period of IHx. Splenectomy abolished the phenomenon. Plasma epinephrine and norepinephrine levels during hypoxia were comparable in IHx and NxC groups, but the epinephrine-induced [Hb] increase was larger in IHx. The alpha1- and alpha2-adrenoreceptor blockade (phentolamine) and alpha2-blockade (yohimbine) abolished the [Hb] increase of IHx rats. Conversely, alpha2-receptor stimulation (oxymetazoline) increased [Hb] during normoxia in IHx but not in NxC. In conclusion, this IHx protocol results in reversible [Hb] increases during hypoxia via splenic contraction mediated by increased alpha2-adrenoreceptor response. This may protect O2 supply during hypoxia without the cardiovascular burden of polycythemia during normoxia.

Adrenergic alpha-1 Receptor Agonists↗

The novel analgesic compound OT-7100 (5-n-butyl-7-(3,4,5-trimethoxybenzoylamino)pyrazolo[1,5-a]pyrimid ine) attenuates mechanical nociceptive responses in animal models of acute and peripheral neuropathic hyperalgesia.

We investigated the effects of OT-7100, a novel analgesic compound (5-n-butyl-7-(3,4,5-trimethoxybenzoylamino)pyrazolo[1,5-a]pyrimidi ne), on prostaglandin E2 biosynthesis in vitro, acute hyperalgesia induced by yeast and substance P in rats and hyperalgesia in rats with a chronic constriction injury to the sciatic nerve (Bennett model), which is a model for peripheral neuropathic pain. OT-7100 did not inhibit prostaglandin E2 biosynthesis at 10(-8)-10(-4) M. Single oral doses of 3 and 10 mg/kg OT-7100 were effective on the hyperalgesia induced by yeast. Single oral doses of 0.1, 0.3, 1 and 3 mg/kg OT-7100 were effective on the hyperalgesia induced by substance P in which indomethacin had no effect. Repeated oral administration of OT-7100 (10 and 30 mg/kg) was effective in normalizing the mechanical nociceptive threshold in the injured paw without affecting the nociceptive threshold in the uninjured paw in the Bennett model. Indomethacin had no effect in this model. While amitriptyline (10 and 30 mg/kg) and clonazepam (3 and 10 mg/kg) significantly normalized the nociceptive threshold in the injured paw, they also increased the nociceptive threshold in the uninjured paw. These results suggest that OT-7100 is a new type of analgesic with the effect of normalizing the nociceptive threshold in peripheral neuropathic hyperalgesia.

Amitriptyline↗

[Long-term prognosis of patients with initial cerebral thrombosis and the MRI findings].

To clarify the relationship between long-term prognosis of patients with stroke and their MRI findings, 103 patients with initial cerebral thrombosis, who survived more than three months after the ictus, were studied for five years. The mean age of 98 patients (T group), who were followed up completely, was 73.1 years-old and 65 were men. The age-matched controls consisted of two groups: 65 subjects, who had hypertension and/or diabetes without a history of stroke (R group), and 85 subjects, who had any hypertension, diabetes and stroke (N group). MRI findings were divided into six categories: 1) types of causative lesion, 2) grades of periventricular hyperintensity (none, rims/caps, patchy, diffuse PVH), 3) number of spotty lesions, 4) presence of silent infarction. 5) ventricular dilatation, and 6) extents of brain atrophy. Types of causative lesion were subdivided into 3 subtypes; infarction of the perforating artery territory (P type), infarction of the cortical artery territory (C type), and brainstem infarction (B type). The presence of vascular risks and dementia, and the extent of activity of daily living (ADL) were assessed. The P, C, and B types were identified by MRI in 46, 36, and 16 of the T group, respectively. Motor impairment, dementia, and an ADL status of complete dependence at discharge were also seen in 84, 44, and 22, respectively. In the T group, 33 patients died during five years, which resulted in a cumulative mortality rate of 33.7% and an annual mortality rate of 8.2%. Based on log-rank analysis, the survival rate of the T group revealed was significantly lower than those of the R and N groups. The recurrent rate in the T group (annual stroke recurrence rate was 4.0%) was higher than in the R and N groups, but stroke recurrence was not the cause of death and two thirds of deaths were due to aspiration pneumonia and/or asphyxia. Cox hazard regression analysis for death due to respiratory diseases showed that the hazard ratios of infarction, patchy PVH, and more than 4 spotty lesions were 8.87 (p < .001), 0.31 (p = .058), and 0.44 (p = .098), respectively. Compared to the survival group, rates of complete dependence in ADL, dementia, and brain atrophy were significantly higher in the death group with low incidences of the P type and patchy PVH, which indicated small vessel disease. These findings suggested that in patients with cerebral thrombosis, even in the chronic phase, care should be taken to prevent pneumonia and/or asphyxia due to bulbar palsy. Furthermore, no MRI findings were distinct predictors of long-term prognosis, although infarction based on the small vessel disease had rather good outcome in terms of respiratory disease.

Aged↗

[Clinical significance of serum lipoprotein(a) concentration in the elderly with regard to vascular complications and long-term prognosis].

To elucidate the clinical significance of lipoprotein(a) [Lp(a)] in the elderly, 48 patients with Lp(a) of 35 mg/dl or more [the high Lp(a) group] and 97 patients with Lp(a) less than 20 mg/dl [the low Lp(a) group] were enrolled to investigate their outcome for five years. At registration, they were all examined by brain computerized tomography (CT) for cerebrovascular diseases, B-mode ultrasonography (US) for carotid lesions, ECG for myocardial ischemia, and Doppler arteriography for the ankle pressure index (API). They were followed up completely to study survival rates, cause of death, and morbidity rates of vascular events, including occurrence of stroke, myocardial infarction, and aortic aneurysm as well as progression of the stage in arteriosclerosis obliterans. The mean age of the high Lp(a) group was 78.1, whereas that of the low Lp(a) group was 76.8. Baseline clinical findings revealed no difference in age or gender between the two groups, although a previous history of stroke, abnormal CT and US findings, and low API were more frequent in the high Lp(a) group than in the low Lp(a) group. In the high Lp(a) group [vs. the low Lp(a) group], 18 patients (vs. 21 patients) died within five years, which resulted in a cumulative mortality rate of 37.5% (vs. 21.6%) and an annual mortality rate of 9.4% (vs. 4.8%). Based on log-rank analysis, the survival rate of the high Lp(a) group was significantly lower than that of the low Lp(a) group. The most common causes of death were vascular events and pneumonia, more than half of them were aspiration pneumonia complicated with stroke. Ten patients in the high Lp(a) group had vascular events (vs. 8 patients). The morbidity rate of vascular events, most of which were cerebral infarction, was higher in the high Lp(a) group (annual morbidity rate 5.5%) than in the low Lp(a) group (1.8%). These findings suggested that serum Lp(a) concentration, genetically determined and remaining consistent throughout life, had influenced vascular wall damage over a long time with age, therefore, a high Lp(a) level might promote atherothrombosis. In the elderly, therefore, high Lp(a) level, resulting in symptomatic vascular lesions with organ dysfunction, is a distinct and independent poor prognostic risk factor.

Aged↗

[Five elderly patients with cerebral infarction seen during a heat wave].

Five elderly patients (> or = 65 y) with cerebral infarction induced by dehydration during a heat wave were described to clarify the relationship between dehydration and stroke in the aged. When the daily maximum temperature exceeded 30 degrees C every day for two weeks, 6 patients with acute stroke came to our hospital. Five of them were patients with cerebral infarction aged 73-89 (the elderly group) and one was a 52-year-old woman with putaminal hemorrhage. As control groups, patients with ischemic stroke during the period 4 weeks before and after, but excluding the heat wave period, which consisted of an elderly control group (n = 7) and a young control group (n = 5), were also studied retrospectively with regard to clinical findings and neuroimaging. The incidence of cerebral infarction in the elderly group was higher in the heat wave period among all three groups. Atherothrombotic, lacunar, and cardioembolic infarctions were seen in 1, 2 and 2 cases, respectively. The onset in the elderly group was characteristic as all occurred before noon and were related to exercise. Physical examination at arrival revealed decreased skin turgor and dry tongue. A high BUN/creatinine ratio (> or = 25) and elevated fibrinogen (> 400 mg/dl) was frequently noted, although high hematocrit (> or = 45) was not seen. According to clinical findings, dehydration was diagnosed and they were infused with fluid, resulting in the improvement of skin turgor and tongue moisture. These findings indicated that dehydration due to excess perspiration due to the heat wave induced cerebral infarction in the elderly. It suggests that water intake on awakening in summer is important to prevent dehydration and ischemic stroke because elderly people are especially susceptible to those conditions in the morning.

Aged↗

[Brain computerized tomographic and ultrasonographic findings in patients with asymptomatic carotid bruits].

This study was conducted to clarify brain and carotid lesions in patients with asymptomatic carotid bruits and their characteristics. We studied 37 patients with carotid bruits, who had various diseases other than stroke and were all neurologically normal, using by brain computerized tomography (CT) and ultrasonography (US). On CT, localized low density areas (LDAs) and their distribution were assessed, as well as the grade of periventricular lucency (PVL). Carotid lesions on US were classified into 3 categories: plaque (locally thickened intima-media complex of 2.1 mm or more in thickness), stenosis (narrowed lumen between 50% and 90% of the linearly measured diameter), and occlusion (severely narrowed lumen more than 90%). Ankle pressure index (API) less than 0.9 was defined as low. Mean age was 73.2 years-old and 28 of them were men. Bruits were heard bilaterally in 15 patients. CT findings showed LDA in 13 patients (35%) and severe PVL in 12 patients (32%). Twenty-three LDAs (13 in the left hemisphere and 10 in the right hemisphere) were seen and all were considered to be infarctions. Nineteen LDAs, 13 of them seen in the basal ganglia, were lacunae. Another 3 LDAs were seen in the watershed zone between the middle and posterior cerebral arteries, whereas the remaining one was a small cortical infarction in the left premotor area in the middle cerebral artery territory. Ultrasonography showed carotid lesions in 65 of 74 carotid arteries (plaque in 28, stenosis in 26, and occlusion in 11) and low API in 18 of 37 patients. Compared with patients with normal CT finding, the frequency of hypertension (92% vs 50%) and ischemic heart disease (69% vs 29%) was significantly high in 13 patients with silent infarction, although there was no difference in US findings. In the hemisphere ipsilateral to the carotid with bruits, which was frequently stenotic, the frequency of infarction was similar to that in the hemisphere ipsilateral to the carotid with no bruit. Regression analysis revealed that hypertension significantly correlated with the presence of cerebral infarction. These findings indicated that incidence of infarction in the elderly patients with asymptomatic carotid bruits was high and was associated with hypertension and advanced atherosclerosis in many organs, including the carotid and peripheral arteries. The reason for the lack of symptoms was considered to be that most of the infarctions were lacunae and located in the basal ganglia, although infarction did not significantly correlate with bruits or carotid lesions.

Aged↗

[Clinical study of transurethral electrovaporization of the prostate for benign prostatic hyperplasia].

We report our clinical experience of transurethral electrovaporization of the prostate (TUVP) for benign prostatic hyperplasia (BPH) using VaporTrode developed by Circon ACMI. From April to November 1995, we treated 22 patients with symptomatic BPH with TUVP. The mean I-PSS decreased significantly, from 21.8 at baseline to 8.1, 4.5, 4.3 and 5.4 at 1, 3, 6 and 12 months after operation, respectively. The mean QOL index also decreased significantly, from 5.2 to 1.5, 1.0, 1.3 and 1.3. The mean peak flow rate increased significantly, from 9.0 preoperatively to 17.4, 17.7, 20.8 and 16.5 ml/sec at 1, 3, 6 and 12 months after TUVP, respectively. The mean prostate volume decreased significantly, from 41.5 to 22.9, 18.6, 18.8 and 19.9 ml. The mean residual urine decreased significantly, from 90.0 to 17.6, 20.6, 24.1 and 9.4 ml. As for overall efficacy, the rate of excellent and good cases at 1, 3, 6 and 12 months was 77.3, 95.5, 95.0 and 84.2%, respectively. No serious complications were observed. Our clinical results suggest that TUVP using VaporTrode has several potential advantages including good efficacy, minimal morbidity and lower cost compared with other less invasive procedures, and may become the useful way of surgical treatment for BPH.

Aged↗

[Is hippocampal atrophy a specific change for Alzheimer's disease?].

Although detection of hippocampal atrophy has been proposed for the diagnosis of Alzheimer's disease (AD), atrophic changes in MRI can be found in other dementia diseases. This study was undertaken to determine whether hippocampal atrophy was a specific change for AD. Coronal T 1-weighted images were performed in 36 patients with AD, 40 patients with non-AD including vascular dementia, frontemporal dementia, Parkinson's disease with dementia, dementia with Lewy bodies, progressive supranuclear palsy, and normal pressure hydrocephalus, 9 patients with age-associated memory impairment (AAMI), and 24 control subjects. Hippocampal atrophy was graded subjectively on a 5-point scale. Scores of hippocampal atrophy for AD (2.11 +/- 0.95) and non-AD (1.80 +/- 0.91) were significantly higher than those for controls (0.79 +/- 0.72). Scores for AD were also significantly higher than those for AAMI (1.11 +/- 0.160), but no difference was found between AD and non-AD. These results suggest that hippocampal atrophy is not a specific marker for AD and appears to be a common phenomenon in dementia syndromes.

Aged↗

Protein kinase C-dependent regulation of Na+/Ca2+ exchanger isoforms NCX1 and NCX3 does not require their direct phosphorylation.

We compared the phosphorylation-dependent regulation of three mammalian Na+/Ca2+ exchanger isoforms (NCX1-NCX3) expressed in CCL39 fibroblasts that have little endogenous activity. Na+i-dependent 45Ca2+ uptake into NCX1- or NCX3-expressing cells, but not that into NCX2-expressing cells, was significantly enhanced by phorbol 12-myristate 13-acetate (PMA) or platelet-derived growth factor-BB, which was abolished by pretreatment of cells with calphostin C or a prior long exposure to PMA. This suggests that NCX1 or NCX3, but not NCX2, is stimulated by a pathway involving protein kinase C (PKC). Immunoprecipitation experiments using [32P]orthophosphate-labeled cells revealed that both NCX2 and NCX3 proteins were phosphorylated to a much lesser extent than the NCX1 protein in unstimulated cells and that the extent of phosphorylation was not increased by treatment with PKC activators, although NCX1 phosphorylation was enhanced significantly. Using site-directed mutagenesis, we identified three phosphorylation sites in the NCX1 protein in the PMA-stimulated cells to be Ser-249, Ser-250, and Ser-357 with Ser-250 being predominantly phosphorylated. We found that the NCX1 mutant with these serine residues substituted with alanine still maintained a normal response to PMA. In contrast, the NCX1 or NCX3 mutant, with the large central cytoplasmic loop deleted, lost the responsiveness to PMA. These results suggest that the PKC-dependent regulation of NCX1 or NCX3 requires the central cytoplasmic loop but does not require the direct phosphorylation of the exchanger.

Amino Acid Sequence↗