Tuberculosis preventive treatment opportunities at antiretroviral therapy initiation and follow-up visits.

SETTING: Twenty-two clinics providing HIV care and treatment in Botswana where tuberculosis (TB) and HIV comorbidity is as high as 49%. OBJECTIVES: To assess eligibility of TB preventive treatment (TPT) at antiretroviral therapy (ART) initiation and at four follow-up visits (FUVs), and to describe the TB prevalence and associated factors at baseline and yield of TB diagnoses at each FUV. DESIGN: A prospective study of routinely collected data on people living with HIV (PLHIV) enrolled into care for the Xpert® MTB/RIF Package Rollout Evaluation Study between 2012 and 2015. RESULTS: Of 6041 PLHIV initiating ART, eligibility for TPT was 69% (4177/6041) at baseline and 93% (5408/5815); 95% (5234/5514); 96% (4869/5079); and 97% (3925/4055) at FUV1, FUV2, FUV3, and FUV4, respectively. TB prevalence at baseline was 11% and 2%, 3%, 3% and 6% at each subsequent FUV. At baseline, independent risk factors for prevalent TB were CD4 <200 cells/mm3 (aOR = 1.4, P = 0.030); anemia (aOR = 2.39, P < 0.001); cough (aOR = 11.21, P < 0.001); fever (aOR = 2.15, P = 0.001); and weight loss (aOR = 2.60, P = 0.002). CONCLUSION: Eligibility for TPT initiation is higher at visits post-ART initiation, while most cases of active TB were identified at ART initiation. Missed opportunities for TB further compromises TB control effort among PLHIV in Botswana.

MARCO DE REFERENCIA: Veintidós consultorios que prestan atención y tratamiento relacionados con la infección por el virus de la inmunodeficiencia humana (VIH) en Botswana, donde la comorbilidad por tuberculosis (TB) e infección por el VIH puede alcanzar 49%. OBJETIVOS: Evaluar los criterios para recibir el tratamiento preventivo de la TB (TPT) durante las consultas de iniciación y seguimiento del tratamiento antirretrovírico (TAR) y describir la prevalencia de TB y los factores asociados en el momento del inicio y el rendimiento del diagnóstico de TB en cada cita de seguimiento del TAR. MÉTODO: Fue este un estudio prospectivo de los datos obtenidos sistemáticamente en las personas con infección por el VIH (PLHIV), inscritas en la atención para el estudio de evaluación del despliegue de la prueba Xpert® MTB/RIF del 2012 al 2015. RESULTADOS: De los 6041 PLHIV que iniciaron el TAR, 69% (4177/6041) cumplía los criterios para recibir el TPT al comienzo; 93% (5408/5815) en la primera consulta de seguimiento; 95% (5234/5514) en la segunda; 96% (4869/5079) en la tercera; y 97% (3925/4075) en la cuarta cita de seguimiento. La prevalencia inicial de TB fue 11% y durante el seguimiento fue 2%, 3%, 3% y 6%, respectivamente. Al comienzo del TAR, los factores de riesgo independientes de diagnóstico de TB fueron una cifra de linfocitos CD4 <200 células/mm3 (aOR 1,4; P = 0,030), la anemia (aOR 2,39; P < 0,001), la tos (aOR 11,21; P = <0,001), la fiebre (aOR 2,15; P = 0,001) y la pérdida de peso (aOR 2,60; P = 0,002). CONCLUSIÓN: Los pacientes cumplen las condiciones para recibir el TPT con mayor frecuencia en las consultas posteriores al comienzo del TAR, pero la mayoría de los casos de TB activa se detecta al iniciarlo. Las oportunidades desaprovechadas para detectar casos de TB dificultan aún más el control de esta enfermedad en las PLHIV en Botswana.

Improving sputum collection processes to increase tuberculosis case finding among HIV-positive persons in Botswana.

SETTING: Twenty-two human immunodeficiency virus (HIV) clinics in Botswana. OBJECTIVE: To compare sputum collection rates, sputum quality and volume, and tuberculosis (TB) diagnosis rates before and after field efforts to improve sputum collection among individuals newly diagnosed with HIV with TB symptoms. DESIGN: Newly diagnosed individuals living with HIV attending 22 HIV clinics in Botswana were screened for TB from August 2012 to March 2014. Starting in May 2013, a field intervention composed of the introduction of a tracking log for presumed TB patients, and patient instructions and sputum induction to improve sputum collection rates was implemented. RESULTS: Prior to the intervention, sputum collection rates were 44.1% (384/870). Subsequently, sputum collection increased to 58.3% (579/993) (P < 0.001). Sputum quality and volume also improved. Although rates of TB diagnosis increased from 9.7% (84/870) to 12.5% (120/993), this difference was not significant (P = 0.143). CONCLUSION: Sputum collection rates among presumptive TB cases, as well as sputum quality and volume improved after implementation of the field intervention. To improve sputum collection rates, efforts at the program level should be ongoing.

Treatment outcomes, diagnostic and therapeutic impact: Xpert vs. smear. A systematic review and meta-analysis.

BACKGROUND: Compared with smear microscopy, Xpert® MTB/RIF has the potential to reduce delays in tuberculosis (TB) diagnosis and treatment initiation, and improve treatment outcomes. We reviewed publications comparing treatment outcomes of drug-susceptible TB patients diagnosed using Xpert vs. smear. METHODS: Citations (2000-2016) reporting treatment outcomes of patients diagnosed using Xpert compared with smear were selected from PubMed, Scopus and conference abstracts. We conducted a systematic review and meta-analysis. Favorable (cured, completed) and unfavorable (failure, death, loss to follow-up) outcomes were pooled for meta-analysis; we also reviewed the number of TB cases diagnosed, time to treatment and empiric treatment. The Mantel-Haenszel method with a fixed-effect model was used; I² was calculated to measure heterogeneity. RESULTS: From 13 citations, 43 594 TB patients were included and 4825 were with known TB treatment outcome. From the pooled analysis, an unfavorable outcomes among those diagnosed using Xpert compared with smear was 20.2%, 541/2675 vs. 21.9%, 470/2150 (risk ratio 0.92, 95%CI 0.82-1.02). Statistical heterogeneity was low (I² = 0.0%, P = 0.910). Compared with smear, Xpert was reported to be superior in increasing the number of TB patients diagnosed (2/9 citations), increasing bacteriologically confirmed TB (7/9 citations), reducing empiric treatment (3/5 citations), reducing time to diagnosis (2/3 citations), and reducing time to treatment initiation (1/5 citations). CONCLUSIONS: Xpert implementation showed no discernible impact on treatment outcomes compared with conventional smear despite reduced time to diagnosis, time to treatment or reduced level of empiric treatment. Further research is required to learn more about gaps in the existing health system.

Tuberculosis in human immunodeficiency virus-infected children starting antiretroviral therapy in Côte d'Ivoire.

SETTING: In Côte d'Ivoire, more than 2000 human immunodeficiency virus (HIV) infected children aged <15 years were started on antiretroviral therapy (ART) during 2004-2008. OBJECTIVES: To estimate tuberculosis (TB) incidence and determinants among ART enrollees. DESIGN: A nationally representative retrospective cohort study among 2110 children starting ART during 2004-2008 at 29 facilities. RESULTS: At ART initiation, the median age was 5.1 years; 82% had World Health Organization Stage III/IV, median CD4% was 11%, 42% were severely undernourished (weight-for-age Z-score [WAZ] <-3), and 150 (7%) were taking anti-tuberculosis treatment. Documentation of TB screening before ART declined from 63% to 46% during 2004-2008. Children taking anti-tuberculosis treatment at ART enrollment had a lower median CD4% (9.0% vs. 11.0%, P = 0.037) and a higher prevalence of WAZ <-3 (59% vs. 40%, P < 0.001). Among children considered TB-free at ART enrollment, TB incidence was 6.28/100 child-years during days 0-90 of ART, declining to 0.56/100 child-years after 180 days. Children with one unit higher WAZ at ART enrollment had 13% lower TB incidence (adjusted HR 0.87, 95%CI 0.77-1.00, P= 0.047). CONCLUSIONS: Ensuring clinician compliance with TB screening before ART and ensuring earlier ART initiation before children suffer from advanced HIV disease and nutritional compromise might reduce TB morbidity during ART.

Piloting the use of personal digital assistants for tuberculosis and human immunodeficiency virus surveillance, Kenya, 2007.

SETTING: Improved documentation of human immunodeficiency virus (HIV) testing and care among tuberculosis (TB) patients is needed to strengthen TB-HIV programs. In 2007, Kenya piloted the use of personal digital assistants (PDAs) instead of paper registers to collect TB-HIV surveillance data from TB clinics. OBJECTIVE: To evaluate the acceptability, data quality and usefulness of PDAs. DESIGN: We interviewed four of 31 district coordinators who collected data in PDAs for patients initiating TB treatment from April to June 2007. In 10 of 93 clinics, we randomly selected patient records for comparison with corresponding records in paper registers or PDAs. Using Cochran-Mantel-Haenszel tests, we compared missing data proportions in paper registers with PDAs. We evaluated PDA usefulness by analyzing PDA data from all 93 clinics. RESULTS: PDAs were well accepted. Patient records were more frequently missing (28/97 vs. 1/112, P < 0.001) and data fields more frequently incomplete (148/1449 vs. 167/2331, P = 0.03) in PDAs compared with paper registers. PDAs, however, facilitated clinic-level analyses: 48/93 (52%) clinics were not reaching the targets of testing >or=80% of TB patients for HIV, and 8 (9%) clinics were providing <80% of TB-HIV co-infected patients with cotrimoxazole (CTX). CONCLUSION: PDAs had high rates of missing data but helped identify clinics that were undertesting for HIV or underprescribing CTX.

Store-operated Ca(2+) inflow in Reuber hepatoma cells is inhibited by voltage-operated Ca(2+) channel antagonists and, in contrast to freshly isolated hepatocytes, does not require a pertussis toxin-sensitive trimeric GTP-binding protein.

The treatment of H4-IIE cells (an immortalised liver cell line derived from the Reuber rat hepatoma) with thapsigargin, 2, 5-di-(tert-butyl)-1,4-benzohydroquinone, cyclopiazonic acid, or pretreatment with EGTA, stimulated Ca(2+) inflow (assayed using intracellular fluo-3 and a Ca(2+) add-back protocol). No stimulation of Mn(2+) inflow by thapsigargin was detected. Thapsigargin-stimulated Ca(2+) inflow was inhibited by Gd(3+) (maximal inhibition at 2 microM Gd(3+)), the imidazole derivative SK&F 96365, and by relatively high concentrations of the voltage-operated Ca(2+) channel antagonists, verapamil, nifedipine, nicardipine and the novel dihydropyridine analogues AN406 and AN1043. The calmodulin antagonists W7, W13 and calmidazolium also inhibited thapsigargin-induced Ca(2+) inflow and release of Ca(2+) from intracellular stores. No inhibition of either Ca(2+) inflow or Ca(2+) release was observed with calmodulin antagonist KN62. Substantial inhibition of Ca(2+) inflow by calmidazolium was only observed when the inhibitor was added before thapsigargin. Pretreatment of H4-IIE cells with pertussis toxin, or treatment with brefeldin A, did not inhibit thapsigargin-stimulated Ca(2+) inflow. Compared with freshly isolated rat hepatocytes, H4-IIE cells exhibited a more diffuse actin cytoskeleton, and a more granular arrangement of the endoplasmic reticulum (ER). In contrast to freshly isolated hepatocytes, the arrangement of the ER in H4-IIE cells was not affected by pertussis toxin treatment. Western blot analysis of lysates of freshly isolated rat hepatocytes revealed two forms of G(i2(alpha)) with apparent molecular weights of 41 and 43 kDa. Analysis of H4-IIE cell lysates showed only the 41 kDa form of G(i2(alpha)) and substantially less total G(i2(alpha)) than that present in rat hepatocytes. It is concluded that H4-IIE cells possess store-operated Ca(2+) channels which do not require calmodulin for activation and exhibit properties similar to those in freshly isolated rat hepatocytes, including susceptibility to inhibition by relatively high concentrations of voltage-operated Ca(2+) channel antagonists. In contrast to rat hepatocytes, SOCs in H4-IIE cells do not require G(i2(alpha)) for activation. Possible explanations for differences in the requirement for G(i2(alpha)) in the activation of Ca(2+) inflow are briefly discussed.

Evidence that the TRP-1 protein is unlikely to account for store-operated Ca2+ inflow in Xenopus laevis oocytes.

The role of the TRP-1 protein, an animal cell homologue of the Drosophila transient receptor potential Ca2+ channel, in store-operated Ca2+ inflow in Xenopus laevis oocytes was investigated. A strategy involving RT-PCR and 3' and 5' rapid amplification of cDNA ends (RACE) was used to confirm and extend previous knowledge of the nucleotide and predicted amino acid sequences of Xenopus TRP-1 (xTRP-1). The predicted amino acid sequence was used to prepare an anti-TRP-l polyclonal antibody which detected the endogenous oocyte xTRP-1 protein and the human TRPC-1 protein expressed in Xenopus oocytes. Ca2+ inflow (measured using fura-2) initiated by 3-deoxy-3-fluoroinositol 1,4,5-trisphosphate (InsP3F) or lysophosphatidic acid (LPA) was completely inhibited by low concentrations of lanthanides (IC50 = 0.5 microM), indicating that InsP3F and LPA principally activate store-operated Ca2+ channels (SOCs). Antisense cRNA or antisense oligodeoxynucleotides, based on different regions of the xTRP-1 cDNA sequence, when injected into Xenopus oocytes, did not inhibit InsP3F-, LPA- or thapsigargin-stimulated Ca2+ inflow. Oocytes expressing the hTRPC-1 protein, which is 96% similar to xTRP-1, exhibited no detectable enhancement of either basal or InsP3F-stimulated Ca2+ inflow and only a very small enhancement of LPA-stimulated Ca2+ in-flow compared with control oocytes. It is concluded that the endogenous xTRP-1 protein is unlikely to be responsible for Ca2+ inflow through the previously-characterised Ca2+ -specific SOCs which are found in Xenopus oocytes. It is considered that xTRP-1 is likely to be a receptor-activated non-selective cation channel such as the channel activated by maitotoxin.

Expression of Drosophila trpl cRNA in Xenopus laevis oocytes leads to the appearance of a Ca2+ channel activated by Ca2+ and calmodulin, and by guanosine 5'[gamma-thio]triphosphate.

The effects of expression of the Drosophila melanogaster Trpl protein, which is thought to encode a putative Ca2+ channel [Phillips, Bull and Kelly (1992) Neuron 8, 631-642], on divalent cation inflow in Xenopus laevis oocytes were investigated. The addition of extracellular Ca2+ ([Ca2+]0) to oocytes injected with trpl cRNA and to mock-injected controls, both loaded with the fluorescent Ca2+ indicator fluo-3, induced a rapid initial and a slower sustained rate of increase in fluorescence, which were designated the initial and sustained rates of Ca2+ inflow respectively. Compared with mock-injected oocytes, trpl-cRNA-injected oocytes exhibited a higher resting cytoplasmic free Ca2+ concentration ([Ca2+]i), and higher initial and sustained rates of Ca2+ inflow in the basal (no agonist) states. The basal rate of Ca2+ inflow in trpl-cRNA-injected oocytes increased with (1) an increase in the time elapsed between injection of trpl cRNA and the measurement of Ca2+ inflow, (2) an increase in the amount of trpl cRNA injected and (3) an increase in [Ca2+]0. Gd3+ inhibited the trpl cRNA-induced basal rate of Ca2+ inflow, with a concentration of approx. 5 microM Gd3+ giving half-maximal inhibition. Expression of trpl cRNA also caused an increase in the basal rate of Mn2+ inflow. The increases in resting [Ca2+]1 and in the basal rate of Ca2+ inflow induced by expression of trpl cRNA were inhibited by the calmodulin inhibitors W13, calmodazolium and peptide (281-309) of (Ca2+ and calmodulin)-dependent protein kinase II. A low concentration of exogenous calmodulin (introduced by microinjection) activated, and a higher concentration inhibited, the trpl cRNA-induced increase in basal rate of Ca2+ inflow. The action of the high concentration of exogenous calmodulin was reversed by W13 and calmodazolium. When rates of Ca2+ inflow in trpl-cRNA-injected oocytes were compared with those in mock-injected oocytes, the guanosine 5'-[beta-thio]diphosphate-stimulated rate was greater, the onset of thapsigargin-stimulated initial rate somewhat delayed and the inositol 1,4,5-trisphosphate-stimulated initial rate markedly inhibited. It is concluded that (1) the divalent cation channel activity of the Drosophila Trpl protein can be detected in Xenopus oocytes: (2) in the environment of the Xenopus oocyte the Trpl channel admits some Mn2+ as well as Ca2+, is activated by cytoplasmic free Ca2+ (through endogenous calmodulin) and by a trimeric GTP-binding regulatory protein, but does not appear to be activated by depletion of Ca2+ in the endoplasmic reticulum; and (3) expression of the Trpl protein inhibits the process by which the release of Ca2+ from intracellular stores activates endogenous store-activated Ca2+ channels.

Injection of rat hepatocyte poly(A)+ RNA to Xenopus laevis oocytes leads to expression of a constitutively-active divalent cation channel distinguishable from endogenous receptor-activated channels.

The expression of hepatocyte plasma membrane receptor-activated divalent cation channels in immature (stages V and VI) Xenopus laevis oocytes and the properties which allow these channels to be distinguished from endogenous receptor-activated divalent cation channels were investigated. Divalent cation inflow to oocytes housed in a multiwell plate was measured using the fluorescent dyes Fluo-3 and Fura-2. In control oocytes, ionomycin, cholera toxin, thapsigargin, 3-fluoro-inositol 1,4,5-trisphosphate (InsP3F) and guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) stimulated Ca2+ and Mn2+ inflow following addition of these ions to the oocytes. Ionomycin-, cholera-toxin-, thapsigargin- and InsP3F-stimulated Ca2+ inflow was inhibited by Gd3+ (half maximal inhibition at less thari 5 microM Gd3+ for InsP3F-stimulated Ca2+ inflow). GTP gamma S-stimulated Ca2+ inflow was insensitive to 50 microM Gd3+ and to SK&F 96365. These results indicate that at least three types of endogenous receptor-activated Ca2+ channels can be detected in Xenopus oocytes using Ca(2+)-sensitive fluorescent dyes: lanthanide-sensitive divalent cation channels activated by intracellular Ca2+ store depletion, lanthanide-sensitive divalent cation channels activated by cholera toxin, and lanthanide-insensitive divalent cation channels activated by an unknown trimeric G-protein. Oocytes microinjected with rat hepatocyte poly(A)+ RNA exhibited greater rates of Ca2+ and Mn2+ inflow in the basal (no agonist) state, greater rates of Ca2+ inflow in the presence of vasopressin or InsP3F and greater rates of Ba2+ inflow in the presence of InsP3F, when compared with "mock"-injected oocytes. In poly(A)+ RNA-injected oocytes, vasopressin- and InsP3F-stimulated Ca2+ inflow, but not basal Ca2+ inflow, was inhibited by Gd3+. It is concluded that at least one type of hepatocyte plasma membrane divalent cation channel, which admits Mn2+ as well as Ca2+ and is lanthanide-insensitive, can be expressed and detected in Xenopus oocytes.

Evidence that neomycin inhibits plasma membrane Ca2+ inflow in isolated hepatocytes.

The effects of neomycin on Ca2+ fluxes and inositol polyphosphates in hepatocytes were investigated since it has been proposed that this antibiotic inhibits inositol 1,4,5-triphosphate formation in fibroblasts [D. H. Carney, D. L. Scott, E. A. Gordon and E. F. LaBelle, Cell 42, 479 (1985)]. In hepatocytes incubated at 1.3 mM extracellular Ca2+ (Ca2+o) neomycin (2 mM) inhibited 45Ca2+ exchange both in the presence or absence of vasopressin. At 1.3 mM Ca2+o, but not at higher concentrations of Ca2+o, the antibiotic (2 mM) inhibited the increase in glycogen phosphorylase a activity observed at late but not at early times after addition of vasopressin. The antibiotic also inhibited the increase in phosphorylase activity caused by the subsequent addition of 1.3 mM Ca2+o to cells previously incubated in the presence of vasopressin and in the absence of added Ca2+o. The concentration of the antibiotic (2 mM) which gave half-maximal inhibition of phosphorylase activation by vasopressin had no effect on the activation of phosphorylase by glucagon or the release of Ca2+ from intracellular stores induced by vasopressin. At a concentration of 10 mM, neomycin caused a 50% inhibition of the formation of [3H]inositol polyphosphates induced by vasopressin. It is concluded that neomycin, at concentrations which inhibit phosphoinositide-specific phospholipase C in other types of cells inhibits the inflow of Ca2+ across the plasma membrane but does not inhibit inositol trisphosphate formation in hepatocytes.

Evidence that a pertussis-toxin-sensitive substrate is involved in the stimulation by epidermal growth factor and vasopressin of plasma-membrane Ca2+ inflow in hepatocytes.

1. In hepatocytes, epidermal growth factor (EFG) (a) increased the rate of 45Ca2+ exchange in cells incubated at 1.3 mM extracellular Ca2+, (b) increased the activity of glycogen phosphorylase a and the intracellular free Ca2+ concentration (measured with quin2) in a process dependent on the concentration of extracellular Ca2+, and (c) enhanced the increase in glycogen phosphorylase activity which follows the addition of Ca2+ to cells previously incubated in the absence of Ca2+. It is concluded that EGF stimulates plasma-membrane Ca2+ inflow. 2. The effects of the combination of EGF and vasopressin on the rate of 45Ca2+ exchange and on the rate of increase in glycogen phosphorylase activity were the same as those of vasopressin alone. 3. The amount of 45Ca2+ released by EGF from internal stores was about 30% of that released by vasopressin. No detectable increase in [3H]inositol mono-, bis- or tris-phosphate was observed after the addition of EGF to cells labelled with myo-[3H]inositol. 4. In hepatocytes isolated from rats treated with pertussis toxin, the effects of EGF and vasopressin on phosphorylase activity (measured at 1.3 mM-Ca2+) and on the rate of Ca2+ inflow (measured with quin2) were markedly decreased compared with those in normal cells. 5. Treatment with pertussis toxin did not impair the ability of vasopressin to release Ca2+ from internal stores, but decreased vasopressin-stimulated [3H]inositol polyphosphate formation by 50%. 6. It is concluded that the mechanism(s) by which vasopressin and EGF stimulate plasma-membrane Ca2+-inflow transporters in hepatocytes involves a GTP-binding regulatory protein sensitive to pertussis toxin, and does not require an increase in the concentration of inositol trisphosphate comparable with that which induces the release of Ca2+ from the endoplasmic reticulum.

Effects of energy deprivation and hydrogen peroxide on contraction and myoplasmic free calcium concentrations in isolated myocardial muscle cells.

The effect of energy deprivation and H2O2 on the contraction, shape, and intracellular free Ca2+ concentration of myocardial muscle cells was investigated using suspensions of freshly isolated, electrically stimulated rat ventricle heart cells. The mitochondrial uncoupling agent carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used to decrease the rate of ATP synthesis. At 0.9 mM extracellular Ca2+, CCCP (0.25 microM) reduced the number of contracting cells by 50% after 5 min, and the number of rod-shaped cells by 40% after 10 min. The effects of CCCP were associated with a substantial decrease in measured cellular ATP concentrations. The deleterious effect of exposure of myocytes to CCCP for periods of up to 5 min was enhanced by an increase in the extracellular Ca2+ concentration, but markedly reduced in the absence of electrical stimulation. Verapamil protected myocytes from the deleterious effects of CCCP during the first 5 min but not at later times. In the presence of 46 mM extracellular K+, CCCP caused a marked increase in the myoplasmic free Ca2+ concentration (measured using quin2). This effect was inhibited by verapamil and was not observed in the absence of K+-induced depolarization. Exposure of myocytes to H2O2 (0.5 mM) caused a substantial decrease both in the number of cells which exhibited normal end-to-end synchronous contraction and in the total number of cells which contracted either partially or fully. The effects of H2O2 were more pronounced at higher concentrations of the peroxide, with longer times of exposure to the agent, and at higher concentrations of extracellular Ca2+, and were partially reversed by dimethyl sulfoxide. The results indicate that both ATP deprivation and H2O2, possibly through the generation of free radicals, cause substantial and rapid damage to cardiac myocytes and induce the movement of additional Ca2+ across the sarcolemma to the myoplasm. In the case of ATP deprivation, this initially occurs through voltage-operated channels.

Effects of verapamil, dibutyryl cyclic AMP, and extracellular calcium on intracellular free calcium concentrations in myocytes isolated from rat ventricles.

The concentration of free calcium in the myoplasm of myocytes isolated from rat ventricles was measured using quin2. Incubation of myocytes with the acetoxymethylester of quin2 (50 mumol.litre-1) for 45 min was required to give effective loading with quin2. At 1 mmol.litre-1 extracellular calcium free calcium was 210(17) (n = 5) nmol.litre-1. Depolarisation of myocytes by addition of 40 mmol.litre-1 potassium caused a threefold increase in free calcium. Increases in extracellular calcium from 0.25 to 2.0 mmol.litre-1 caused a twofold increase in free calcium in polarised (resting) myocytes and a 2.5-fold increase in cells depolarised with potassium. The ability of verapamil to inhibit the electrically stimulated contraction of myocytes was dependent on the stimulation voltage. Half-maximal inhibition was given by 0.7 and 2 mumol.litre-1 verapamil at 50 and 100 V stimulation respectively. High concentrations of verapamil completely inhibited potassium induced increases in the fluorescence of quin2 loaded cells. Half-maximal inhibition was observed at 0.5 mumol.litre-1 verapamil. The calcium agonist CGP 28392 enhanced potassium induced fluorescence of quin2 loaded cells. Dibutyryl cyclic adenosine monophosphate and adrenaline increased the proportion of rod shaped cells that contracted in response to stimulation by low, but not high, voltage. Dibutyryl cyclic AMP caused a threefold increase in the potassium induced increase in free calcium. It is concluded that verapamil decreases and that calcium agonists, dibutyryl cyclic AMP, and increases in extracellular calcium increase free calcium, as monitored by intracellular quin2.

Effect of extracellular Ca2+ on plasma membrane Ca2+ inflow and cytoplasmic free Ca2+ in isolated hepatocytes.

An initial rapid phase and a subsequent slow phase of 45Ca2+ uptake were observed following the addition of 45Ca2+ to Ca2+-deprived hepatocytes. The magnitude of the rapid phase increased 15-fold over the range 0.1-11 mM extracellular Ca2+ (Ca2+o) and was a linear function of [Ca2+]o. The increases in the rate of 45Ca2+ uptake were accompanied by only small increases in the intracellular free Ca2+ concentration. In cells made permeable to Ca2+ by treatment with saponin, the rate of 45Ca2+ uptake (measured at free Ca2+ concentrations equal to those in the cytoplasm of intact cells) increased as the concentration of saponin increased from 1.4 to 2.5 micrograms per mg wet weight cells. Rates of 45Ca2+ uptake by cells permeabilized with an optimal concentration of saponin were comparable with those of intact cells incubated at physiological [Ca2+o], but were substantially lower than those for intact cells incubated at high [Ca2+o]. It is concluded that Ca2+ which enters the hepatocyte across the plasma membrane is rapidly removed by binding and transport to intracellular sites and by the plasma membrane (Ca2+ + Mg2+)-ATPase and the plasma membrane Ca2+ inflow transporter is not readily saturated with Ca2+o.

Studies with verapamil and nifedipine provide evidence for the presence in the liver cell plasma membrane of two types of Ca2+ inflow transporter which are dissimilar to potential-operated Ca2+ channels.

The addition of 500 microM verapamil or nifedipine to isolated hepatocytes incubated in the presence of 1.3 mM Ca2+ caused 20% inhibition of Ca2+ inflow as measured by the initial rate of 45Ca2+ exchange. No stimulation of 45Ca2+ exchange was observed in the presence of the Ca2+ agonist CGP 28392. An increase in the concentration of extracellular K+ from 6 to 60 mM (to depolarize the plasma membrane) increased the initial rate of 45Ca2+ exchange by 30%. In the presence of 60 mM K+, 400 microM verapamil inhibited the initiate rate of 45Ca2+ exchange by 50%. Verapamil and nifedipine completely inhibited vasopressin-induced Ca2+ inflow as determined by measurement of the initial rate of 45Ca2+ exchange and of glycogen phosphorylase a activity. This effect of verapamil was completely reversed by increasing the extracellular concentration of Ca2+. The concentrations of Ca2+ antagonist which gave 50% inhibition of vasopressin- or K+-stimulated Ca2+ inflow were in the range 50-100 microM, about 50-fold greater than the concentration which gave 50% inhibition of the beating of electrically-stimulated myocardial muscle cells. In the absence of vasopressin, verapamil caused a transient increase in glycogen phosphorylase a activity by a process which is largely independent of Ca2+. It is concluded that verapamil and nifedipine inhibit the transport of Ca2+ across the hepatocyte plasma membrane through a putative Ca2+ transporter which is activated by vasopressin and which differs in nature from potential-operated Ca2+ channels in excitable cells and from the Ca2+ transporter present in hepatocytes in the absence of hormone.

Intraspinal narcotic analgesia in the treatment of chronic pain.

The results of intraspinal narcotic analgesia (INA) in 43 patients with chronic nonmalignant pain syndromes are reviewed. A protocol has been established to improve proper patient selection and includes three phases of study. Most of the patients have had INA for 2 years now. In those patients qualifying for continuous delivery systems (CDS), 65% had good to excellent relief of pain while 34% were considered failures for a variety of reasons. Apparent tolerance development in many of the patients was, in fact, due to technical problems with the epidural catheter instead.

Myelographic defect on the side opposite the leg pain. A case report with an explanation of mechanism of action.

An explanation for leg pain on the opposite side of the myelographic defect in one case is reported. Disc herniation was located superior to the exit of the root and thus displaced the dura and compressed the root on the opposite side against the pedicle producing contralateral leg pain.

Chronic spinal arachnoiditis. A postoperative syndrome that may signal its onset.

Chronic lumbar radiculopathy following spinal surgery is reported, in which 7 of 25 patients reviewed developed a postoperative syndrome immediately after their original surgery. Later, sometimes years later, all 7 patients developed severe chronic spinal arachnoiditis. This syndrome was characterized by transient violent spasms in the legs, muscle cramps, increased radicular pain, and often fever and chills. The recognition of this syndrome and a proposed method of treatment is discussed.