The Mexican consensus on irritable bowel syndrome. / Consenso mexicano sobre el síndrome de intestino irritable.

BACKGROUND: Since the publication in 2009 of the Guidelines on the Diagnosis and Treatment of Irritable Bowel Syndrome of the Asociación Mexicana de Gastroenterología (2009 Guidelines), there have been significant advances in our knowledge of the epidemiology, pathophysiology, diagnosis, and treatment of this disease. AIMS: To present a consensus review of the most current knowledge of IBS, updating the 2009 Guidelines by incorporating new internationally published scientific evidence, with a special interest in Mexican studies. METHODS: The PubMed literature from January 2009 to March 2015 was reviewed and complemented through a manual search. Articles in English and Spanish were included and preference was given to consensuses, guidelines, systematic reviews, and meta-analyses. Statements referring to the different aspects of the disease were formulated and voted upon by 24 gastroenterologists employing the Delphi method. Once a consensus on each statement was reached, the quality of evidence and strength of recommendation were determined through the GRADE system. RESULTS: Forty-eight statements were formulated, updating the information on IBS and adding the complementary data that did not appear in the 2009 Guidelines regarding the importance of exercise and diet, diagnostic strategies, and current therapy alternatives that were analyzed with more stringent scientific vigor or that emerged within the last 5 years. CONCLUSIONS: We present herein a consensus review of the most relevant advances in the study of IBS, updating and complementing the 2009 Guidelines. Several studies conducted in Mexico were included.

Hypoxic induction of T-type Ca(2+) channels in rat cardiac myocytes: role of HIF-1α and RhoA/ROCK signalling.

T-type Ca(2+) channels are expressed in the ventricular myocytes of the fetal and perinatal heart, but are normally downregulated as development progresses. Interestingly, however, these channels are re-expressed in adult cardiomyocytes under pathological conditions. We investigated low voltage-activated T-type Ca(2+) channel regulation in hypoxia in rat cardiomyocytes. Molecular studies revealed that hypoxia induces the upregulation of Cav 3.2 mRNA, whereas Cav 3.1 mRNA is not significantly altered. The effect of hypoxia on Cav 3.2 mRNA was time- and dose-dependent, and required hypoxia inducible factor-1α (HIF-1α) stabilization. Patch-clamp recordings confirmed that T-type Ca(2+) channel currents were upregulated in hypoxic conditions, and the addition of 50 µm NiCl2 (a T-type channel blocker) demonstrated that the Cav 3.2 channel is responsible for this upregulation. This increase in current density was not accompanied by significant changes in the Cav 3.2 channel electrophysiological properties. The small monomeric G-protein RhoA and its effector Rho-associated kinase I (ROCKI), which are known to play important roles in cardiovascular physiology, were also upregulated in neonatal rat ventricular myocytes subjected to hypoxia. Pharmacological experiments indicated that both proteins were involved in the observed upregulation of the Cav 3.2 channel and the stabilization of HIF-1α that occurred in response to hypoxia. These results suggest a possible role for Cav 3.2 channels in the increased probability of developing arrhythmias observed in ischaemic situations, and in the pathogenesis of diseases associated with hypoxic Ca(2+) overload.

Effects of region of birth, educational level and age on late presentation among men who have sex with men newly diagnosed with HIV in a network of STI/HIV counselling and testing clinics in Spain.

This paper analyses late presentation (LP) of HIV infection, and its determinants, among men who have sex with men (MSM) in Spain, newly diagnosed with HIV (2003-2011) in 15 sexually transmitted infection/HIV counselling and testing clinics. LP was defined as <350 CD4 cells/µL or AIDS. In total, 3,081 MSM were included (2,499 having CD4/AIDS); overall LP was 25.3%. LP was higher in men older than 34 years, those not previously HIV-tested (adjusted odds ratio (aOR):3.1; 95% confidence intervals (CI):2.3-4.2) , and those tested > 12 months before diagnosis (12-24 months (aOR:1.4; 95% CI:1.0-2.0); > 24 months (aOR:2.2; 95% CI:1.7-3.0)). LP was less likely in MSM reporting a known HIV-infected partner as infection source or symptoms compatible with acute retroviral syndrome. 'Region of birth' interacted with 'educational level' and 'steady partner as infection source': only African and Latin-American MSM with low educational level were more likely to present late; Latin-American men attributing their infection to steady partner, but no other MSM, had LP more frequently. In Spain, HIV testing among MSM should be promoted, especially those > 34 years old and migrants with low educational level. The current recommendation that MSM be tested at least once a year is appropriate.

Trends in HIV testing, prevalence among first-time testers, and incidence in most-at-risk populations in Spain: the EPI-VIH Study, 2000 to 2009.

During 2000 to 2009, data on people undergoing HIV testing and on those newly diagnosed with HIV were collected in a network of 20 Spanish clinics specialising in sexually transmitted infections and/or HIV testing and counselling. The number of tests performed, overall and disaggregated by different variables, was obtained. HIV prevalence among first-time testers and HIV incidence among repeat testers were calculated. To evaluate trends, joinpoint regression models were fitted. In total, 236,939 HIV tests were performed for 165,745 individuals. Overall HIV prevalence among persons seeking HIV testing was 2.5% (95% CI: 2.4 to 2.6). Prevalence was highest in male sex workers who had sex with other men (19.0% (95% CI: 16.7 to 21.4)) and was lowest in female sex workers (0.8% (95% CI: 0.7 to 0.9)). Significant trends in prevalence were observed in men who have sex with men (MSM) (increasing) and heterosexual individuals (decreasing). The incidence analysis included 30,679 persons, 64,104 person-years (py) of follow-up and 642 seroconversions. The overall incidence rate (IR) was 1.0/100 py (95% CI: 0.9/100 to 1.1/100). Incidence was significantly higher in men and transgender females than in women (1.8/100 py (95% CI: 1.6 to 1.9), 1.2/100 py (95% CI: 0.5 to 2.8) and 0.1/100 py (95% CI: 0.09 to 0.2) respectively) and increased with age until 35­39 years. IRs in MSM and people who inject drugs were significantly greater than in heterosexual individuals (2.5/100 py (95% CI: 2.3 to 2.7), 1.6/100 py (95% CI: 1.1 to 2.2) and 0.1/100 py (95% CI: 0.09 to 0.2) respectively), and an upward trend was observed in MSM. Our results call for HIV prevention to be reinforced in MSM and transgender women in Spain.

[Prevalence of irritable bowel syndrome in Veracruz City, Mexico: a community-based survey]. / Prevalencia del síndrome de intestino irritable en población abierta de la ciudad de Veracruz, México.

BACKGROUND: Irritable bowel syndrome (IBS) is recognized as the most frequent functional digestive disorder around the world. In Latin America and Mexico there are few studies in order to demonstrate its real prevalence in general population. AIMS: To determine the prevalence of IBS in general population from Veracruz City Mexico, using the Rome II criteria. MATERIAL AND METHODS: Using basic information given by bureau for planning urban services from Veracruz country, a 10% random population sample was obtained. Subjects between 16-80 years old were interviewed using a questionnaire based on Rome II criteria and a visual analogous scale in order to estimate the negative effect of IBS symptoms on daily activities. RESULTS: We interviewed 459 subjects with a median age of 31.2 +/- 13.6 years old detecting 78 subjects (16.9%) with IBS symptoms: 25 males and 53 females (gender prevalence of 11.3% and 22.1%, respectively). 28.2% of them had IBS with diarrhea, 50% had IBS with constipation and 21.8% alternating bowel movements, diarrhea and constipation. Negative effect of IBS symptoms on daily activities was significant. CONCLUSIONS: The prevalence of IBS in open population was 16.9% according to Rome II criteria, being higher in those older than 35 years old. Constipation was the predominant pattern. Further studies should evaluate associated factors of these findings.

Chemotransduction in the carotid body: K+ current modulated by PO2 in type I chemoreceptor cells.

The ionic currents of carotid body type I cells and their possible involvement in the detection of oxygen tension (Po2) in arterial blood are unknown. The electrical properties of these cells were studied with the whole-cell patch clamp technique, and the hypothesis that ionic conductances can be altered by changes in PO2 was tested. The results show that type I cells have voltage-dependent sodium, calcium, and potassium channels. Sodium and calcium currents were unaffected by a decrease in PO2 from 150 to 10 millimeters of mercury, whereas, with the same experimental protocol, potassium currents were reversibly reduced by 25 to 50 percent. The effect of hypoxia was independent of internal adenosine triphosphate and calcium. Thus, ionic conductances, and particularly the O2-sensitive potassium current, play a key role in the transduction mechanism of arterial chemoreceptors.

HIV/STI co-infection among men who have sex with men in Spain.

In Spain, neither the HIV nor the STI national surveillance systems collect information on HIV/STI co-infection. However, there are two networks based on HIV/STI clinics which gather this data. We describe HIV prevalence in men who have sex with men (MSM) diagnosed with infectious syphilis and/or gonorrhoea in 15 STI clinics; and concurrent diagnoses of STI in MSM newly diagnosed with HIV in 19 HIV/STI clinics. In total, 572 MSM were diagnosed with infectious syphilis and 580 with gonorrhoea during 2005-2007. HIV prevalence among syphilis and gonorrhoea cases was 29.8% and 15.2% respectively. In the multivariate analysis, HIV/syphilis co-infection was associated with being Latin American; having a history of STI; reporting exclusively anal intercourse; and having sex with casual or several types of partners. HIV and gonorrhoea co-infection was associated with age older than 45 years; having no education or only primary education completed; and having a history of STI. In total, 1,462 HIV infections were newly diagnosed among MSM during 2003-2007. Of these, 31.0% were diagnosed with other STI at the same time. Factors associated with STI co-infection among new HIV cases in MSM were being Latin American; and having sex with casual partners or with both steady and casual partners. In Spain, a considerable proportion of MSM are co-infected with HIV and STI.

Dry sliding wear behaviour of ß-type Ti-Nb and Ti-Mo surfaces designed by diffusion treatments for biomedical applications.

The dry sliding wear behaviour of different Ti-Nb and Ti-Mo surfaces was investigated in order to evaluate the role of Nb and Mo ß-stabilizing elements in titanium wear resistance to consider them for biomedical applications. Dry sliding wear tests were performed under unlubricated conditions using a ball-on-plate tribometer (UMT) with reciprocating lineal movement of 1 Hz frequency at different loads (2 and 5 N) and against two counterface materials (alumina and stainless steel) to assess the effect of these parameters on wear. The results indicated an improvement in wear resistance for all the modified Ti surfaces. Metal-on-metal surfaces exhibited higher wear rate than ceramic-on-metal, and higher wear was observed for the more severe conditions. Wear rate values on modified surfaces were between 53% and 96% lower compared to pure Ti tested at 2 N, and up to 79% lower than Ti at 5 N. In both cases the highest wear reduction was observed for Ti-MoNH4Cl surface.

Dexamethasone-induced upregulation of CaV3.2 T-type Ca2+ channels in rat cardiac myocytes.

Glucocorticoids are widely used to treat acute and chronic diseases. Unfortunately, their therapeutic use is associated with severe side effects. Glucocorticoids are known to regulate several ion channels in cardiac myocytes, including voltage-dependent Ca2+ channels. Low-voltage-activated T-type Ca2+ channels are expressed in ventricular myocytes during the fetal and perinatal period, but are practically absent in the adult. However, these channels can be re-expressed in adult cardiomyocytes under some pathological conditions. We have investigated the glucocorticoid regulation of T-type Ca2+ channels in rat cardiomyocytes. Molecular studies revealed that dexamethasone induces the upregulation of CaV3.2 mRNA in neonatal rat ventricular myocytes, whereas CaV3.1 mRNA is only slightly affected. Patch-clamp recordings confirmed that T-type Ca2+ channel currents were upregulated in dexamethasone treated cardiomyocytes, and the addition of 50 µmol/L NiCl2 demonstrated that the CaV3.2 channel is responsible for this upregulation. The effect of dexamethasone on CaV3.2 is mediated by the activation and translocation to the cell nucleus of the glucocorticoid receptor (GR). We have isolated the upstream promoter of the Cacna1h gene and tested its activity in transfected ventricular myocytes. The initial in silico analysis of Cacna1h promoter revealed putative glucocorticoid response elements (GREs). Transcriptional activity assays combined with deletion analyses and chromatin immunoprecipitation assays demonstrated that GR binds to a region a GRE located in -1006/-985 bp of Cacna1h promoter. Importantly, upregulation of the CaV3.2 channel is also observed in vitro in adult rat ventricular myocytes, and in vivo in a rat model of excess of glucocorticoids.

Hierarchy of binding sites for Grb2 and Shc on the epidermal growth factor receptor.

We analyzed the binding site(s) for Grb2 on the epidermal growth factor (EGF) receptor (EGFR), using cell lines overexpressing EGFRs containing various point and deletion mutations in the carboxy-terminal tail. Results of co-immunoprecipitation experiments suggest that phosphotyrosines Y-1068 and Y-1173 mediate the binding of Grb2 to the EGFR. Competition experiments with synthetic phosphopeptides corresponding to known autophosphorylation sites on the EGFR demonstrated that phosphopeptides containing Y-1068, and to a lesser extent Y-1086, were able to inhibit the binding of Grb2 to the EGFR, while a Y-1173 peptide did not. These findings were confirmed by using a dephosphorylation protection assay and by measuring the dissociation constants of Grb2's SH2 domain to tyrosine-phosphorylated peptides, using real-time biospecific interaction analysis (BIAcore). From these studies, we concluded that Grb2 binds directly to the EGFR at Y-1068, to a lesser extent at Y-1086, and indirectly at Y-1173. Since Grb2 also binds Shc after EGF stimulation, we investigated whether Y-1173 is a binding site for the SH2 domain of Shc on the EGFR. Both competition experiments with synthetic phosphopeptides and dephosphorylation protection analysis demonstrated that Y-1173 and Y-992 are major and minor binding sites, respectively, for Shc on the EGFR. However, other phosphorylation sites in the carboxy-terminal tail of the EGFR are able to compensate for the loss of the main binding sites for Shc. These analyses reveal a hierarchy of interactions between Grb2 and Shc with the EGFR and indicate that Grb2 can bind the tyrosine-phosphorylated EGFR directly, as well as indirectly via Shc.

SH2 domains exhibit high-affinity binding to tyrosine-phosphorylated peptides yet also exhibit rapid dissociation and exchange.

src homology 2 (SH2) domains of intracellular signaling molecules such as phospholipase C-gamma and phosphatidylinositol 3'-kinase-associated protein p85 represent recognition motifs for specific phosphotyrosine-containing regions on activated growth factor receptors. The binding of SH2 domains to activated growth factor receptors controls the interaction with signaling molecules and the regulation of their activities. In this report, we describe the kinetic parameters and binding affinities of SH2 domains of p85 toward short phosphotyrosine-containing peptides with the amino acid sequence motif YMXM, derived from a major insulin receptor substrate, IRS-1, by using real time biospecific interaction analysis (BIAcore). Associations were specific and of very high affinity, with dissociation constants of 0.3 to 3 nM, between phosphopeptides and the two separate SH2 domains contained within p85. Nonphosphorylated peptides showed no measurable binding, and the interactions were specific for the primary sequence very close to the phosphotyrosine residue. Moreover, the interactions between phosphopeptides and SH2 domains of other signaling molecules were of much lower affinity. Interestingly, the binding of the SH2 domains to the tyrosine-phosphorylated peptides was of high affinity as a result of a very high on rate, of 3 x 10(7) to 40 x 10(7)/M/s; at the same time, the rate of dissociation, of 0.11 to 0.19/s, was rapid, allowing for rapid exchange of associating proteins with the tyrosine phosphorylation sites.

Ligand binding to heparan sulfate proteoglycans induces their aggregation and distribution along actin cytoskeleton.

Cell surface heparan sulfate proteoglycans (HSPGs) participate in molecular events that regulate cell adhesion, migration, and proliferation. The present study demonstrates that soluble heparin-binding proteins or cross-linking antibodies induce the aggregation of cell surface HSPGs and their distribution along underlying actin filaments. Immunofluorescence and confocal microscopy and immunogold and electron microscopy indicate that, in the absence of ligands, HSPGs are irregularly distributed on the fibroblast cell surface, without any apparent codistribution with the actin cytoskeleton. In the presence of ligand (lipoprotein lipase) or antibodies against heparan sulfate, HSPGs aggregate and colocalize with the actin cytoskeleton. Triton X-100 extraction and immunoelectron microscopy have demonstrated that in this condition HSPGs were clustered and associated with the actin filaments. Crosslinking experiments that use biotinylated lipoprotein lipase have revealed three major proteoglycans as binding sites at the fibroblast cell surface. These cross-linked proteoglycans appeared in the Triton X-100 insoluble fraction. Platinum/carbon replicas of the fibroblast surface incubated either with lipoprotein lipase or antiheparan sulfate showed large aggregates of HSPGs regularly distributed along cytoplasmic fibers. Quantification of the spacing between HSPGs by confocal microscopy confirmed that the nonrandom distribution of HSPG aggregates along the actin cytoskeleton was induced by ligand binding. When cells were incubated either with lipoprotein lipase or antibodies against heparan sulfate, the distance between immunofluorescence spots was uniform. In contrast, the spacing between HSPGs on fixed cells not incubated with ligand was more variable. This highly organized spatial relationship between actin and proteoglycans suggests that cortical actin filaments could organize the molecular machinery involved in signal transduction and molecular movements on the cell surface that are triggered by heparin-binding proteins.

Expression of discoidin domain receptor 1 during mouse brain development follows the progress of myelination.

Discoidin domain receptor 1 is a tyrosine kinase receptor expressed in a variety of tissues including the brain. This study describes mRNA and protein expression of discoidin domain receptor 1 in mouse brain during development and provides new insights into its role during gliogenesis and neurogenesis. We performed in situ hybridization for discoidin domain receptor 1 in mouse brains at embryonic day 18, postnatal days 5, 9, 15, 21 and adulthood and observed a diffuse pattern in the proliferative areas during embryogenesis. From postnatal day 5 onwards, a defined cellular expression pattern of discoidin domain receptor 1 was observed, mainly located in white matter tracts and following a spatio-temporal pattern that overlapped the progress of myelination. Next, we performed double-labeling reactions (in situ hybridization followed by immunohistochemistry) that confirmed that discoidin domain receptor 1 was expressed by mature oligodendrocytes. We observed that cells positive for discoidin domain receptor 1 also expressed carnosine and anti-adenomatous polyposis coli, two mature oligodendrocyte markers. Based on the localization of discoidin domain receptor 1 specifically in the white matter fiber tracts during postnatal development, we suggest that discoidin domain receptor 1 participates in the development and maintenance of the myelin sheath.

Inactivation of glutathione S-transferase P1 gene by promoter hypermethylation in human neoplasia.

Glutathione S-transferases (GSTs) are a family of isoenzymes that play an important role in protecting cells from cytotoxic and carcinogenic agents. The pi-class GST has been associated with preneoplastic and neoplastic changes. Recently, it has been reported that regulatory sequences near the GSTP1 gene, which encodes the human pi-class GST, are commonly hypermethylated in prostatic carcinomas. In the present study, we studied more than 300 primary human tumors originating in other organs for aberrant methylation of GSTP1 using methylation-specific PCR. GSTP1 hypermethylation was most frequent in breast and renal carcinoma, showing aberrant methylation in 30 and 20% of the cases, respectively. Other tumor types showed promoter methylation only rarely or not at all. Hypermethylation of GSTP1 was associated with loss of expression demonstrated by immunohistochemistry. Our results suggest that aberrant methylation of GSTP1 may contribute to the carcinogenetic process in breast and renal carcinomas.

Immunological properties of rat phosphoglycerate mutase isozymes.

In mammalian tissues three phosphoglycerate mutase (D-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1) isozymes result from the homo-dimeric and hetero-dimeric combinations of two subunits (types M and B). Whereas rabbit antisera against type M subunit (purified from rat muscle) and against type BB isozyme (purified from rat brain) possessed a high degree of specificity, both antisera reacted with type BB and MM isozymes, as demonstrated by immunoneutralization and ELISA. Both the M subunit and B subunit were more immunoreactive than their respective dimeric isozymes. Subunits type M and B may possess common antigenic determinants, and some of these determinants may be sterically hindered in their dimeric structures.

Properties of calcium and potassium currents of clonal adrenocortical cells.

The ionic currents of clonal Y-1 adrenocortical cells were studied using the whole-cell variant of the patch-clamp technique. These cells had two major current components: a large outward current carried by K ions, and a small inward Ca current. The Ca current depended on the activity of two populations of Ca channels, slow (SD) and fast (FD) deactivating, that could be separated by their different closing time constants (at -80 mV, SD, 3.8 ms, and FD, 0.13 ms). These two kinds of channels also differed in (a) activation threshold (SD, approximately -50 mV; FD, approximately -20 mV), (b) half-maximal activation (SD, between -15 and -10 mV; FD between +10 and +15 mV), and (c) inactivation time course (SD, fast; FD, slow). The total amplitude of the Ca current and the proportion of SD and FD channels varied from cell to cell. The amplitude of the K current was strongly dependent on the internal [Ca2+] and was almost abolished when internal [Ca2+] was less than 0.001 microM. The K current appeared to be independent, or only slightly dependent, of Ca influx. With an internal [Ca2+] of 0.1 microM, the activation threshold was -20 mV, and at +40 mV the half-time of activation was 9 ms. With 73 mM external K the closing time constant at -70 mV was approximately 3 ms. The outward current was also modulated by internal pH and Mg. At a constant pCa gamma a decrease of pH reduced the current amplitude, whereas the activation kinetics were not much altered. Removal of internal Mg produced a drastic decrease in the amplitude of the Ca-activated K current. It was also found that with internal [Ca2+] over 0.1 microM the K current underwent a time-dependent transformation characterized by a large increase in amplitude and in activation kinetics.

Ionic currents in dispersed chemoreceptor cells of the mammalian carotid body.

Ionic currents of enzymatically dispersed type I and type II cells of the carotid body have been studied using the whole cell variant of the patch-clamp technique. Type II cells only have a tiny, slowly activating outward potassium current. By contrast, in every type I chemoreceptor cell studied we found (a) sodium, (b) calcium, and (c) potassium currents. (a) The sodium current has a fast activation time course and an activation threshold at approximately -40 mV. At all voltages inactivation follows a single exponential time course. The time constant of inactivation is 0.67 ms at 0 mV. Half steady state inactivation occurs at a membrane potential of approximately -50 mV. (b) The calcium current is almost totally abolished when most of the external calcium is replaced by magnesium. The activation threshold of this current is at approximately -40 mV and at 0 mV it reaches a peak amplitude in 6-8 ms. The calcium current inactivates very slowly and only decreases to 27% of the maximal value at the end of 300-ms pulses to 40 mV. The calcium current was about two times larger when barium ions were used as charge carriers instead of calcium ions. Barium ions also shifted 15-20 mV toward negative voltages the conductance vs. voltage curve. Deactivation kinetics of the calcium current follows a biphasic time course well fitted by the sum of two exponentials. At -80 mV the slow component has a time constant of 1.3 +/- 0.4 ms whereas the fast component, with an amplitude about 20 times larger than the slow component, has a time constant of 0.16 +/- 0.03 ms. These results suggest that type I cells have predominantly fast deactivating calcium channels. The slow component of the tails may represent the activity of a small population of slowly deactivating calcium channels, although other possibilities are considered. (c) Potassium current seems to be mainly due to the activity of voltage-dependent potassium channels, but a small percentage of calcium-activated channels may also exist. This current activates slowly, reaches a peak amplitude in 5-10 ms, and thereafter slowly inactivates. Inactivation is almost complete in 250-300 ms. The potassium current is reversibly blocked by tetraethylammonium. Under current-clamp conditions type I cells can spontaneously fire large action potentials. These results indicate that type I cells are excitable and have a variety of ionic conductances. We suggest a possible participation of these conductances in chemoreception.

Low pO2 selectively inhibits K channel activity in chemoreceptor cells of the mammalian carotid body.

The hypothesis that changes in environmental O2 tension (pO2) could affect the ionic conductances of dissociated type I cells of the carotid body was tested. Cells were subjected to whole-cell patch clamp and ionic currents were recorded in a control solution with normal pO2 (pO2 = 150 mmHg) and 3-5 min after exposure to the same solution with a lower pO2. Na and Ca currents were unaffected by lowering pO2 to 10 mmHg, however, in all cells studied (n = 42) exposure to hypoxia produced a reversible reduction of the K current. In 14 cells exposed to a pO2 of 10 mmHg peak K current amplitude decreased to 35 +/- 8% of the control value. The effect of low pO2 was independent of the internal Ca2+ concentration and was observed in the absence of internal exogenous nucleotides. Inhibition of K channel activity by hypoxia is a graded phenomenon and in the range between 70 and 120 mmHg, which includes normal pO2 values in arterial blood, it is directly correlated with pO2 levels. Low pO2 appeared to slow down the activation time course of the K current but deactivation kinetics seemed to be unaltered. Type I cells subjected to current clamp generate large Na- and Ca-dependent action potentials repetitively. Exposure to low pO2 produces a 4-10 mV increase in the action potential amplitude and a faster depolarization rate of pacemaker potentials, which leads to an increase in the firing frequency. Repolarization rate of individual action potentials is, however, unaffected, or slightly increased. The selective inhibition of K channel activity by low pO2 is a phenomenon without precedents in the literature that explains the chemoreceptive properties of type I cells. The nature of the interaction of molecular O2 with the K channel protein is unknown, however, it is argued that a hemoglobin-like O2 sensor, perhaps coupled to a G protein, could be involved.

Oxygen sensing by ion channels and chemotransduction in single glomus cells.

We have monitored cytosolic [Ca2+] and dopamine release in intact fura-2-loaded glomus cells with microfluoroimetry and a polarized carbon fiber electrode. Exposure to low PO2 produced a rise of cytosolic [Ca2+] with two distinguishable phases: an initial period (with PO2 values between 150 and approximately 70 mm Hg) during which the increase of [Ca2+] is very small and never exceeds 150-200 nM, and a second phase (with PO2 below approximately 70 mm Hg) characterized by a sharp rise of cytosolic [Ca2+]. Secretion occurs once cytosolic [Ca2+] reaches a threshold value of 180 +/- 43 nM. The results demonstrate a characteristic relationship between PO2 and transmitter secretion at the cellular level that is comparable with the relation described for the input (O2 tension)output (afferent neural discharges) variables in the carotid body. Thus, the properties of single glomus cells can explain the sensory functions of the entire organ. In whole-cell, patch-clamped cells, we have found that in addition to O2-sensitive K+ channels, there are Ca2+ channels whose activity is also regulated by PO2. Ca2+ channel activity is inhibited by hpoxia, although in a strongly voltage-dependent manner. The average hypoxic inhibition of the calcium current in 30% +/- 10% at -20 mV but only 2% +/- 2% at +30 mV. The differential inhibition of K+ and Ca2+ channels by hypoxia helps to explain why the secretory response of the cells is displaced toward PO2 values (below approximately 70 mm Hg) within the range of those normally existing in arterial blood. These data provide a conceptual framework for understanding the cellular mechanisms of O2 chemotransduction in the carotid body.

Role of Sp1 in the induction of p27 gene expression in vascular smooth muscle cells in vitro and after balloon angioplasty.

-The abnormal proliferation of vascular smooth muscle cells (VSMCs) plays an important role in atherosclerosis and restenosis. Although several studies have implicated the growth inhibitory protein p27(Kip1) (p27) in the control of myocyte growth and hypertrophy, little is known about the molecular mechanisms that regulate p27 expression in the cardiovascular system. In the present study, we demonstrate the interaction of the transcription factor Sp1 with 2 GC-rich sequences within the p27 promoter in cultured VSMCs. Importantly, point mutations that disrupted Sp1 binding markedly reduced p27 promoter activity, demonstrating that Sp1 is required for efficient p27 gene transcription in cultured VSMCs. Because p27 expression is upregulated after balloon angioplasty, we investigated Sp1 expression and activity in control and balloon-injured rat carotid arteries to assess the role of Sp1 as a physiological regulator of p27 expression. Although immunohistochemical analysis disclosed Sp1 protein expression in both control and balloon-injured arteries, a high level of Sp1 DNA-binding activity was found only in response to balloon angioplasty. Collectively, these results demonstrate that Sp1 is essential for maximum p27 promoter activity in VSMCs and suggest that posttranslational induction of Sp1 DNA-binding activity contributes to the induction of p27 expression and VSMC growth arrest at late time points after balloon angioplasty.