Independent association of bone mineral density and trabecular bone score to vertebral fracture in male subjects with chronic obstructive pulmonary disease.

Osteoporosis is a major comorbidity of chronic obstructive pulmonary disease (COPD), but the mechanism of bone fragility is unknown. We demonstrated that trabecular bone score, a parameter of bone quality, was associated with systemic inflammation and was a significant determinant of vertebral fracture independent of bone mineral density. INTRODUCTION: COPD is a major cause of secondary osteoporosis. However, the mechanism of bone fragility is unclear. We previously reported that vertebral fracture was highly prevalent in male COPD patients. To obtain clues to the mechanism of COPD-associated osteoporosis, we attempted to identify determinants of prevalent vertebral fracture in this study. METHODS: In this cross-sectional study, we recruited 61 COPD males and examined pulmonary function, vertebral fractures, bone mineral density (BMD), trabecular bone score (TBS), bone turnover markers, and inflammatory parameters. Determinants of the bone parameters were examined by multivariable analyses. RESULTS: The prevalence of any and grade 2 or 3 fractures was 75.4 and 19.7%, respectively. Osteoporosis and osteopenia defined by BMD were present in 37.7 and 39.3%, respectively. TBS was significantly lower in higher Global Initiative for Chronic Obstructive Lung Disease (GOLD) stages compared to GOLD 1. Multivariable logistic regression analysis revealed that both TBS and BMD were independent determinants of grade 2 or 3 vertebral fractures (OR = 0.271, 95%CI 0.083-0.888, p = 0.031; OR = 0.242, 95%CI 0.075-0.775, p = 0.017) after adjustment for age. Correlates of TBS included age, BMD, high-sensitivity C-reactive protein (hsCRP), pulmonary function parameters, parathyroid hormone, and Tracp-5b. In multivariable regression analysis, hsCRP was the only independent determinant of TBS besides age and BMD. In contrast, independent determinants of BMD included body mass index and, to a lesser extent, 25-hydroxyvitamin D. CONCLUSION: Both BMD and TBS were independently associated with grade 2 or 3 vertebral fracture in COPD male subjects, involving distinct mechanisms. Systemic inflammation, as reflected by increased hsCRP levels, may be involved in deterioration of the trabecular microarchitecture in COPD-associated osteoporosis, whereas BMD decline is most strongly associated with weight loss.

Intracellular Mg2+ influences both open and closed times of a native Ca2+-activated BK channel in cultured human renal proximal tubule cells.

Effects of intracellular Mg2+ on a native Ca(2+)-and voltage-sensitive large-conductance K+ channel in cultured human renal proximal tubule cells were examined with the patch-clamp technique in the inside-out mode. At an intracellular concentration of Ca2+ ([Ca2+](i)) of 10(-5)-10(-4) M, addition of 1-10 mM: Mg2+ increased the open probability (P(o)) of the channel, which shifted the P(o) -membrane potential (V(m)) relationship to the negative voltage direction without causing an appreciable change in the gating charge (Boltzmann constant). However, the Mg(2+)-induced increase in P(o) was suppressed at a relatively low [Ca2+](i) (10(-5.5)-10(-6) M). Dwell-time histograms have revealed that addition of Mg2+ mainly increased P(o) by extending open times at 10(-5) M Ca2+ and extending both open and closed times simultaneously at 10(-5.5) M Ca2+. Since our data showed that raising the [Ca2+](i) from 10(-5) to 10(-4) M increased P(o) mainly by shortening the closed time, extension of the closed time at 10(-5.5) M Ca(2+) would result from the Mg(2+)-inhibited Ca(2+)-dependent activation. At a constant V(m), adding Mg2+ enhanced the sigmoidicity of the P(o)-[Ca2+](i) relationship with an increase in the Hill coefficient. These results suggest that the major action of Mg2+ on this channel is to elevate P(o) by lengthening the open time, while extension of the closed time at a relatively low [Ca2+](i) results from a lowering of the sensitivity to Ca2+ of the channel by Mg2+, which causes the increase in the Hill coefficient.

[Prognosis for the cases after resection of pulmonary metastasis of osteosarcoma].

Osteosarcoma commonly metastasize to the lungs. Sixty-six cases with surgical treatment for pulmonary metastasis of osteosarcomas have been experienced for past 25 years in our hospital. Disease free interval (DFI) and the number of metastatic lesions were assessed in relation to their prognosis. As a result of our assessment, fewer metastasis (3 or less), and longer DFI (1 year or more) had relations to longer post-thoracotomy survival. And the relations between the number of metastatic lesions and DFI were found. Fewer metastasis were associated with longer DFI, and multiple metastasis were associated with short DFI. The cases with fewer metastasis (3 or less) and longer DFI (1 year or more) might have a survival advantage.

Role of nitric oxide in control of light adaptive cone photomechanical movements in retinas of lower vertebrates: a comparative species study.

The possible role of nitric oxide (NO) as a novel light adaptive neuromodulator of cone plasticity (photomechanical movements) in retinae of two contrasting species of fish (freshwater carp and marine bream) and an example of an amphibian (Xenopus laevis) was studied pharmacologically by cytomorphometric measurements. Application of a NO donor [S-nitroso-N-acetyl-d, l-penicillamine] (500-700 microM) to dark-adapted retinae induced contraction of cones with an efficiency (CE) relative to full light adaptation of around 54% in all three species. Pretreatment with a NO scavenger [2-(4-Carboxyphenyl)-4,4,5,5-tetrametylimidazoline-1-oxil-3-oxide] (30-35 microM) produced a consistent significant inhibition of the light adaptation-induced cone contraction (CE = 15-20%). These results strongly suggest the involvement of endogenous NO in the cone contractions that occur in freshwater and marine fish and amphibian retinae as a part of the light adaptation process.

Coronary artery bypass grafting for coronary aneurysms due to Kawasaki disease.

We report a 23-year-old man who underwent coronary artery bypass grafting (CABG) for coronary aneurysms associated with Kawasaki disease using the left internal thoracic artery (LITA) and right gastroepiploic artery (RGEA) after a second myocardial infarction (MI). Preoperatively, this patient showed repetitive occlusion and recanalization of coronary artery flow without coronary stenosis. Indication of bypass surgery in Kawasaki disease is usually associated with stenosis. However, even an aneurysm alone should be an indication of surgery if there is any kind of ischemic event.

Properties of a Ca(2+)-activated large conductance K(+) channel with ATP sensitivity in human renal proximal tubule cells.

The properties of a native Ca(2+)-activated large conductance K(+) channel (BK channel) present in the surface membrane of cultured human renal proximal tubule epithelial cells (RPTECs) were investigated by using the patch-clamp technique. The slope conductance of the BK channel was about 295 pS, and the channel was selective to K(+) over Na(+), with a selectivity ratio of about 12.2. The activity of the channel was almost maximally enhanced by 10(-4 )M or more Ca(2+) in the cytoplasmic surface of the patch membrane and was markedly diminished by reducing the cytoplasmic Ca(2+) to 10(-6) M at the membrane potential of about 0 mV. The depolarization of the patch membrane also enhanced the channel activity, and hyperpolarization lowered it. K(+) channel blockers, Ba(2+) (0.1-1 mM), tetraethylammonium (1 mM), and charybdotoxin (100 nM), were effective for the suppression of channel activity. A significant feature of the K(+) channel was that channel activity maintained by 10(-5)-10(-4 )M Ca(2+) in inside-out patches was inhibited by the addition of ATP (1-10 mM) to the bath solution. ATPgammaS, and a nonhydrolyzable ATP analogue, 5'-adenylylimidodiphosphate (AMP-PNP), also had inhibitory effects on channel activity. However, an inhibitor of ATP-sensitive K(+) channels, glibenclamide (0.1 mM), induced no appreciable change in channel activity from both intra- and extracellular sides. These results suggest that besides the common natures of the BK channel family such as regulation by cytoplasmic Ca(2+) and membrane potential, the BK channel in RPTECs is directly inhibited by intracellular ATP independent of phosphorylation processes and sulfonylurea receptor.

An ATP-regulated and pH-sensitive inwardly rectifying K(+) channel in cultured human proximal tubule cells.

Although renal K(+) channels along the nephron have been explored in various animal species, little is known about the K(+) channels in human proximal tubule cells. Using the patch-clamp technique, we investigated the properties of an inwardly rectifying K(+) channel present in the surface membrane of cultured human proximal tubule cells of normal kidney origin. This channel was the most frequently observed K(+) channel in cell-attached patches, and cytoplasmic ATP was required to maintain channel activity in inside-out patches. Its single channel conductance was about 42 pS for inward currents and 7 pS for outward currents under the symmetrical K(+) condition. The ATP effect on channel activity was dose-dependently stimulatory within a range of 0.1 to 10 mM, and a nonhydrolyzable ATP analog, AMP-PNP (3 mM), had no effect on channel activity in either the presence or absence of ATP (1 mM). The channel activity observed in cell-attached patches was reduced to 30 to 50% of controls by a membrane-permeable nonspecific protein kinase inhibitor, K252a (1 microM), or a potent protein kinase A inhibitor, KT5720 (500 nM). In contrast, a membrane-permeable cAMP analog, 8Br-cAMP (100 microM), induced a twofold increase in channel activity. The addition of a catalytic subunit of protein kinase A (PKA-CS, 100 U/ml) to the bath in inside-out patches stimulated channel activity in the presence of 1 mM ATP. Furthermore, the channel activity maintained with 1 mM ATP in inside-out patches was suppressed by internal acidification and enhanced by alkalization. These results suggest that the activity of the inwardly rectifying K(+) channel in cultured human proximal tubule cells was ATP-dependent and regulated at least in part by cAMP/PKA-mediated phosphorylation processes and intracellular pH.

Dexamethasone induces sodium-dependant vitamin C transporter in a mouse osteoblastic cell line MC3T3-E1.

The regulation of intracellular ascorbic acid (AsA) levels may be under the control of an AsA-specific membrane transporter. The present study investigates AsA uptake and expression of Na-dependent vitamin C transporter (SVCT) mRNA in the mouse osteoblastic cell line, MC3T3-E1. Among eight compounds tested, dexamethasone (Dex) all-trans retinoic acid, transforming growth factor beta, prostaglandin E2 and transferrin significantly and respectively) stimulated the update of AsA into MC3T3-E1 cells. Among these five, Dex was the most active, inducing mSVCT2 mRNA and the uptake of AsA in a time- and concentration-dependant manner. Dex did not induce mSVCT1 mRNA. These results suggest that the Dex-induced stimulation of AsA incorporation into osteoblastic cells is mediated by the induction of mSVCT2. Since Dex reduced alkaline phosphatase activity in MC3T3-E1 cells in our culture conditions, Dex-induced stimulation of AsA incorporation might not be the result of differentiation. Hormone-regulated changes of SVCT expression may have an important role in cell functions.

Expression profiles of voltage-gated Na(+) channel alpha-subunit genes in rat and human prostate cancer cell lines.

BACKGROUND: Voltage-gated Na(+) channel (VGSC) activity has been implicated in prostate cancer (PC) metastasis. Although VGSCs can occur as multiple-subunit assemblies, the alpha-subunits (VGSCalphas) alone can encode functional channels. The VGSCalpha gene(s) responsible for the functional VGSCalpha expression in strongly metastatic PC cell lines is not known. METHODS: Two reverse transcription-PCR (RT-PCR) methods, degenerate primer screening and a novel semi quantitative PCR (SQT-PCR) technique, were used. These methods enabled a detailed qualitative and quantitative investigation of VGSCalpha mRNA expression in rat (MAT-LyLu/AT-2) and human (PC-3/LNCaP) PC cells of markedly different metastatic potential. RESULTS: Expression of eight different VGSCalpha genes (SCN1A-4A, SCN7A-9A, and SCN11A) was determined in the PC cell lines. Most were expressed as multiple splice variants. SQT-PCR results were consistent with a basal level of VGSCalpha mRNA expression occurring in weakly metastatic (AT-2/LNCaP) cells, and this being greatly elevated in cells of stronger metastatic potential (MAT-LyLu/PC-3), primarily due to the elevated expression of the SCN9A gene (also termed PN1/hNe-Na). CONCLUSIONS: (1) Several VGSCalpha genes and their splice variants are expressed similarly in both rat and human PC cell lines. (2) Expression levels are much higher in the strongly metastatic (MAT-LyLu/PC-3) cells. (3) Levels of SCN9A mRNA specifically are predominant in MAT-LyLu and PC-3 cells; thus, SCN9A is highly likely to be the main source of the functional VGSC detected.

[Efficacy of off-pump coronary artery bypass grafting in patients requiring noncardiac operations].

Off-pump coronary artery bypass grafting was performed for patients with concomitant disease requiring noncardiac operations. Eight patients underwent CABG prior to or at the same time of noncardiac operation: lung cancer (2), gastric cancer (2), arteriosclerotic occlusive disease (2), abdominal aortic aneurysm (1), aorto-iliac occlusive disease (1). Of these, there were 6 patients who underwent off-pump CABG. Two patients underwent conventional CABG with extracorporeal circulation. Off-pump CABG was performed through a median sternotomy and small left thoracotomy. One patient received quadruple grafts, and another one received double, and 4 received single grafting. Simultaneous noncardiac operations were carried out in 3 patients (Y-grafting, femoro-femoral bypass, mediastinal lymphnode biopsy). The other 3 patients underwent subsequent operations (axillo-femoro-popliteal bypass, aorto-femoral bypass, subtotal gastrectomy). However, of the 2 patients with conventional CABG, 1 died of multiple metastasis after lobectomy of lung cancer that might have been affected by the extra-corporeal circulation. Off-pump CABG is efficient in patients with concomitant disease requiring noncardiac operations.

Light-adaptive role of nitric oxide in the outer retina of lower vertebrates: a brief review.

The role of nitric oxide (NO) as a novel neurochemical mechanism controlling light adaptation of the outer retina is discussed by considering mainly published results. The emphasis is on the retinae of fishes and amphibia, but some data from the mammalian (rabbit) retinae have also been included for completeness. In the fish retina, application of NO donors in the dark caused light-adaptive photomechanical movements of cones. The normal effect of light adaptation in inducing cone contractions was suppressed by pretreatment of retinae with an NO scavenger. NO donors modulated horizontal cell activity by uncoupling the cells' lateral gap junctional interconnections and enhancing negative feedback to cones, again consistent with a light-adaptive role of NO. Direct evidence for light adaptation-induced release of NO has been obtained in fish (carp) and rabbit retinae. The results strongly suggest that control of retinal light adaptation is, under multiple neurochemical control, with NO and dopamine having an interactive role.

Regulation of inwardly rectifying K(+) channel in cultured opossum proximal tubule cells by protein phosphatases 1 and 2A.

The inwardly rectifying ATP-regulated K(+) channel with an inward conductance of about 90 pS in the surface membrane of cultured opossum kidney proximal tubule (OKP) cells is activated at least in part by protein kinase A (PKA). In this study, we examined the effects of protein serine/threonine phosphatase types 1 (PP-1) and 2A (PP-2A) on activity of the K(+) channel using the patch-clamp technique. In cell-attached patches, channel activity was enhanced by the application of okadaic acid (OA, 1 microM), a membrane-permeable inhibitor of PP-1 and PP-2A, to the bath solution. This enhancement was abolished by the pretreatment of cells with KT5720 (200 nM), a specific inhibitor of PKA. In inside-out patches, channel activity which could be maintained in the presence of ATP (3 mM) in the bath solution was also increased by the addition of OA (1 microM), and the OA-induced increase in channel activity was partially prevented in the presence of KT5720 (200 nM). Direct application of either PP-1 (1 U/ml) or PP-2A (1 U/ml) to the cytoplasmic surface of the patch membrane inhibited channel activity maintained by ATP (3 mM) in inside-out patches. Moreover, channel activity stimulated by PKA (20 nM) in the presence of ATP (3 mM) was also inhibited by the application of either PP-1 (1 U/ml) or PP-2A (1 U/ml). These results indicate that the OA-sensitive protein phosphatase is involved in the regulation of channel activity, and suggest that both PP-1 and PP-2A are candidates responsible for the inhibition of channel activity through dephosphorylation of the PKA-mediated protein phosphorylation.

Goosecoid suppresses cell growth and enhances neuronal differentiation of PC12 cells.

In all vertebrate species, the homeobox gene goosecoid serves as a marker of the Spemann organizer tissue. One function of the organizer is the induction of neural tissue. To investigate the role of goosecoid in neuronal differentiation of mammalian cells, we have introduced goosecoid into PC12 cells. Expression of goosecoid resulted in reduced cell proliferation and enhanced neurite outgrowth in response to NGF. Expression of goosecoid led to a decrease in the percentage of S-phase cells and to upregulation of the expression of the neuron-specific markers MAP-1b and neurofilament-L. Analysis of goosecoid mutants revealed that these effects were independent of either DNA binding or homodimerization of Goosecoid. Coexpression of the N-terminal portion of the ets transcription factor PU.1, a protein that can bind to Goosecoid, repressed neurite outgrowth and rescued the proliferation of PC12 cultures. In contrast, expression of the bHLH transcription factor HES-1 repressed goosecoid-mediated neurite outgrowth without changing the proportion of S-phase cells. These results suggest that goosecoid is involved in neuronal differentiation in two ways, by slowing the cell cycle and stimulating neurite outgrowth, and that these two events are separately regulated.

Low levels of NPM gene expression in UV-sensitive human cell lines.

Nucleophosmin (NPM) is a major nuclear matrix protein associated with neoplastic growth in various cell types. We recently suggested that expression of the NPM gene is involved in an increased resistance to UV irradiation in human cells against the cell-killing effects of UV (mainly 254nm wavelength far-ultraviolet ray) [Y. Higuchi, K. Kita, H. Nakanishi, X-L. Wang, S. Sugaya, H. Tanzawa, H. Yamamori, K. Sugita, A. Yamaura, N. Suzuki, Biochem. Biophys. Res. Commun. 248 (1998) 597-602]. In the present study, expression levels of the NPM gene were examined in human cell lines with a high sensitivity to UV cell-killing. Cockayne syndrome patient-derived cell lines, CSAI and CSBI, and the Xeroderma pigmentosum patient-derived cell line, XP2OS(SV), XP13KY, XP3KA, XP6BE(SV), XP101OS and XP3BR(SV), have been investigated for their NPM mRNA expression with Northern blotting analysis. All of these UV-sensitive cells demonstrated lower expression levels compared with those of normal fibroblast cells, FF, or an UV-resistant cell line, UH(r)-10; quite a lower level of expression in XP205(SV) cells after UV irradiation in contrast to a distinguishable increase in the expression in UV(r)- cells. These results confirmed an intimate correlation between degree of UV sensitivity and expression levels of the NPM gene in human cells.

Distinct mechanisms of transport of ascorbic acid and dehydroascorbic acid in intestinal epithelial cells (IEC-6).

Ascorbic acid (AsA) is an essential nutrient for humans as they lack its biosynthesizing key enzyme. Its absorption mechanism in small intestinal epithelial cells still remains to be resolved. In this study, the transport mechanisms functioning on the uptake of AsA and its oxidized form, dehydroascorbic acid (DHA), were investigated using rat small intestinal epithelial cell line IEC-6. Both AsA and DHA were accumulated in the cells in time- and concentration-dependent manners, but their absorption kinetics were apparently different. The saturability of AsA uptake was shown at a considerably lower concentration in IEC-6 cells as well as other mammalian cells, indicating that this absorption was mediated by a specific transporting carrier. The absorption efficiency of AsA was about 1/5-1/10 that of DHA at the same concentration range and, moreover, the uptake of DHA was almost comparable to that of 2-deoxy-D-glucose, an alternative of glucose. The uptake of AsA was diminished by the removal of sodium ion, but not by the addition of glucose, whereas that of DHA was sodium ion-independent and effectively inhibited by glucose. In addition, phlorizin and cytochalasin B, which are blockers of glucose transporters, interfered the uptake of DHA more efficiently than that of AsA. These results indicate that there are at least two distinct transport systems of vitamin C in rat small intestinal epithelial cells; AsA is transported by a specific transporter and DHA is mainly transported by glucose transporter(s).

Comparable effects of flickering and steady patterns of light adaptation on photomechanical responses of cones in amphibian (Xenopus laevis) retina.

The effects of two distinct patterns of light stimulus, steady and flicker, on cone photomechanical movements (PMMs) in the Xenopus laevis retina were investigated. For both patterns studied, the effects on PMMs were assessed by quantitative analysis of the cone positions in the outer retina. Steady light adaptation was found to be equally effective as flicker in causing cone contractions. This was unlike the situation previously found in the cyprinid fish retina, in which flickering light was significantly more effective than steady. This difference could be related to the light-evoked response characteristics and circuitry of dopaminergic retinal neurones in the two vertebrate classes. The role of dopamine and other possible neuromodulator(s) in light adaptive control of vertebrate retinae is discussed.

Changes in immune function following surgery for esophageal carcinoma.

Changes in immune function due to surgical injury have been well-documented. Immunosuppression is one of the causes of infectious complications leading to organ dysfunction in critical illness. It is not known what kind of surgery in the daily clinical practice causes immunosuppression. Stress response and immune function following surgery for esophageal carcinoma, assuming a highly-stressed operation, were studied and then compared with the stress response and immune function following gastric surgery, a moderately-stressed procedure. Forty patients who underwent esophagectomy and 39 patients receiving gastric operation were studied. The concentrations of serum interleukin-6 (IL-6) were measured preoperatively, at 1, 2, and 6 h, and at 1, 3, and 10 d after operation. Total protein, serum albumin, rapid turnover protein, serum CRP, and cortisol were measured before operation and at 1, 3, 7, and 21 d after operation. ConA- and PHA-stimulated lymphocyte proliferation, IgA, IgG, and IgM were also measured preoperatively, and on 7 and 21 d following surgery. The patients were fed exclusively by total parenteral nutrition (TPN). A striking rise of IL-6 was observed, with a peak in both groups at 1 to 6 h following operation. The peak values were 419+/-30 pg/mL, which was approximately twice as high in the esophagectomy patients as in the gastrectomy patients (195+/-40 pg/mL). CRP and cortisol also increased after operation, and these increases were also significantly greater in the esophagectomy patients. ConA- and PHA-stimulated lymphocyte proliferation decreased significantly 7 d after esophagectomy (P<0.05), but was unchanged in the patients receiving gastrectomy. Suppression of cellular immunity correlated significantly with serum cortisol, and was preceded by a rise in serum IL-6. The IgA, IgG, and IgM levels, however, remained unchanged from their preoperative values throughout the study in both groups. Nutritional status in terms of serum protein, albumin, and rapid turnover protein, decreased postoperatively, but there was no difference between the two groups. It is, therefore, concluded that cell-mediated immunosuppression, preceded by a hyperinflammatory response, is an observable reaction in patients following esophageal surgery, but not in patients undergoing gastric surgery.

Effects of total parenteral nutrition on endotoxin translocation and extent of the stress response in burned rats.

Postburn endotoxin translocation has been well documented. However, the relationship between the secretion of catabolic hormones, degree of endotoxin translocation, and intestinal atrophy has not been previously demonstrated. In this experiment, modulation of the secretion of catabolic hormones according to the route of nutrient administration was examined in burned animals. A total of 55 rats, with and without a burn injury, were orally or parenterally fed. Urinary excretion of epinephrine and norepinephrine (U-EN) of each rat was measured for 48 h after burn injury as an indicator of the stress response. Evaluations of intestinal atrophy and endotoxin contents in the liver and spleen were also done 48 h after burn injury. U-EN after burn injury in rats administered total parenteral nutrition (TPN) was higher than in those fed orally. Endotoxin translocation and intestinal atrophy after thermal injury were also augmented by TPN. A significant positive correlation between U-EN and endotoxin content of the liver, and a negative correlation between U-EN and weight of the intestine, were observed. TPN enhances the stress response after burn injury. An increase in endotoxin translocation and intestinal atrophy by TPN are closely related to enhancement of the stress response.

Myofibrillar protein catabolism is rapidly suppressed following protein feeding.

The immediate response of protein degradation to food intake and the factors for its regulation in rat skeletal muscle were examined. The concentration of N tau-methylhistidine (MeHis) in serum and the rates of MeHis release from isolated soleus and extensor digitorum longus muscles were reduced in the period from 3 to 6 h after refeeding, indicating that the rate of myofibrillar protein degradation in the rat decreased immediately after refeeding. Changes in the serum concentration of insulin and corticosterone were not synchronized with those in the myofibrillar protein degradation. When rats were fed on a protein-free diet, no reduction of serum MeHis concentration or of the rate of MeHis release from isolated muscles after refeeding was apparent. Furthermore, there was a tendency toward suppressing myofibrillar protein degradation with a higher protein content of the diet. These results suggest that the suppression of myofibrillar protein degradation by food intake was regulated by dietary proteins.

Dopamine receptor subtypes expressed in vertebrate (carp and eel) retinae: cloning, sequencing and comparison of five D1-like and three D2-like receptors.

Eight dopamine receptor-like cDNA clones were isolated from the carp (Cyprinus carpio) retina and four dopamine receptor-like cDNA clones were isolated from the European eel (Anguilla anguilla) retina. These cDNA clones show high sequence and structural homology to the known dopamine receptor subtypes. The sequence similarity and phylogenetic analysis revealed that five subtypes (D1A3, D1A4, D1B, D1C and D1X) in the carp retina and four subtypes (D1A1, D1A2, D1B and D1C) in the eel retina are D1-like receptor subtypes, and three (D2, D4A and D4B) in the carp retina are D2-like receptor subtypes; no D2-like receptor was found in the eel. Carp D1A3 and D1A4, carp D4A and D4B, and eel D1A1 and D1A2 are highly homologous pairs of receptors which show significant, domain-specific differences to each other and to their species homologues. The structure of the third cytoplasmic loop in the carp D1X receptor was particularly different from the other D1-like receptors. The implications of these structural differences in terms of dopamine receptor activation and signalling are discussed. It is suggested that the known diverse physiological and pharmacological effects of dopamine on the retinal neurones are likely to be mediated through these multiple receptor subtypes which may be coupled to different signal transduction pathways.