Cutaneous vasodilatation responses synchronize with sweat expulsions

To examine whether cutaneous active vasodilatation is mediated by sudomotor nerve fibres we recorded cutaneous blood flow and sweat rates continuously with laser-Doppler flowmetry and capacitance hygrometry, respectively, from the dorsal and plantar aspects of the foot in 11 male subjects at varying ambient temperatures (T _a) between 22 and 40°C (relative humidity 40%). In a warmer environment (T _a 29–40°C), predominant responses of the blood flow curve from the sole of the foot were transient depressions (negative blood flow responses, NBR), whereas those from the dorsal foot were transient increases (positive blood flow responses, PBR). The PBR on the dorsal foot occurred spontaneously or in response to mental or sensory stimuli, and when PBR did not fuse with each other the rate of PBR was linearly related to tympanic temperature. When dorsal foot sweating was continuous, PBR on the dorsal foot almost entirely synchronized with sweat expulsion. When dorsal foot sweating was intermittent PBR sometimes occurred on the dorsal foot without corresponding sweat expulsions, but these PBR showed a complete correspondence with subthreshold sweat expulsion seen on a methacholine-treated area. The amplitude and the duration of PBR showed a significant linear relationship with the amplitude and the duration of the corresponding sweat expulsion. In a thermoneutral or cooler environment (T _a 22–29°C), PBR occurred on the sole of the foot when mental or sensory stimuli elicited sweating in that area. Thus, PBR occurred when and where sweating appeared. Atropine failed to abolish PBR on the dorsal foot. Blockade of the peroneal nerve eliminated both PBR and NBR on the dorsal foot. The results indicate that an active vasodilatation mechanism is present on the sole of the foot as well as on the dorsal foot, and thus suggest that active vasodilatation is closely related to sudomotor nerve activation.     

Retention of basic electrophysiologic properties by human sweat duct cells in primary culture

Summary The present investigation was undertaken to examine the usefulness of cultured human sweat duct cells for ion transport and related studies in the genetic disease, cystic fibrosis. Electrical properties of cultured duct (CD) cells were compared with electrical properties of microperfused duct (MPD) cells. The resting apical membrane potential (V _a ) of the CD cells was −26.4±0.9 mV,n=158 cells as compared to −24.3±0.6 mV,n=105 of MPD cells. The Na⁺−K⁺ pump inhibitor ouabain, when applied to the apical surface of the CD cells and basolateral surface of MPD cells, depolarized both CD cells (from −28.6±3.6 to −16.8±2.4 mV,n=5) and MPD cells (from −23.8±0.5 mV to −19.5±1.8 mV,n=6). The Na⁺ conductance inhibitor amiloride applied to the apical surface hyperpolarized the apical membrane potentials (V_a) of CD cells and MPD cells by −13.2±1.4 mV,n=43 and −34.3±3.1 mV,n=19), respectively, indicating the presence of amiloride sensitive Na⁺ channels in both groups of cells. However, the amiloride sensitivity of CD cells was dependent on the age of the culture. Cl⁻ substitution at the apical side by the impermeant anion gluconate depolarized the V _a of CD cells and MPD cells by 12.2±0.9 mV,n=32 and 37.9±4.3 mV,n=12, respectively. The effect of β-adrenergic agonist isoproterenol (IPR), was inconsistent. In CD cells, IPR either hyperpolarized (ΔV _a =−8.3±1.2mV,n=5) or depolarized (ΔV _a =8.2±2.3 mV,n=4) or had no effect,n=2. In contrast, most of the MPD cells did not respond to IPR, but three cells had a varied response to IPR. Our results suggest that CD cells, like MPD cells, retain significant Na⁺ and Cl⁻ conductances. CD cells seem to have developed a higher sensitivity to β-adrenergic stimulation in tissue culture as compared to MPD cells. This work was supported by grants from the National Institutes of Health, Bethesda, MD, DK26547, Getty Oil Co., the Gillette Co., Cystic Fibrosis Research Inc., and the U.S. National Cystic Fibrosis Foundation.     

Predicted sweat rates for group water planning in sport: accuracy and application

This study tested the accuracy of a novel, limited-availability web application (H₂Q™) for predicting sweat rates in a variety of sports using estimates of energy expenditure and air temperature only. The application of predictions for group water planning was investigated for soccer match play. Fourteen open literature studies were identified where group sweat rates were reported (n = 20 group means comprising 230 individual observations from 179 athletes) with fidelity. Sports represented included: walking, cycling, swimming, and soccer match play. The accuracy of H₂Q™ sweat rates was tested by comparing to measured group sweat rates using the concordance correlation coefficient (CCC) with 95% confidence interval [CI]. The relative absolute error (RAE) with 95% [CI] was also assessed, whereby the mean absolute error was expressed relative to an acceptance limit of 0.250 L/h. The CCC was 0.98 [0.95, 0.99] and the RAE was 0.449 [0.279, 0.620], indicating that the prediction error was on average 0.112 L/h. The RAE was < 1.0 for 19/20 observations (95%). Drink volumes modeled as a proxy for sweat losses during soccer match play prevented dehydration (< 1% loss of body mass). The H₂Q™ web application demonstrated high group sweat prediction accuracy for the variety of sports activities tested. Water planning for soccer match play suggests the feasibility of easily and accurately predicting sweat rates to plan group water needs and promote optimal hydration in training and/or competition.     

Increased levels of NAD(P)H: quinone oxidoreductase 1 (NQO1) in pancreatic tissues from smokers and pancreatic adenocarcinomas: A potential biomarker of early damage in the pancreas

NAD(P)H:quinone oxidoreductase 1 (NQO1) is elevated in several human tumors. This study was conducted to determine whether increased levels of NQO1 expression also occur in human pancreatic tumor tissue, and to compare expression levels in nontumorous tissue from smokers with those in nonsmokers. The expression of NQO1 was examined in pancreatic tissue samples from 82 human donors. These samples included normal (n = 20), smokers (n = 25), pancreatitis (n = 7), and adenocarcinomas of the pancreas (n = 30). Genotyping for the C609T polymorphism in NQO1 by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis was also performed. Polymorphic variants were confirmed by automatic sequencing. Higher levels of NQO1 expression were demonstrated in pancreatic adenocarcinomas (0.831 ± 0.021) compared to those in nontumorous tissues from nonsmokers (0.139 ± 0.024). These high levels were also found in smokers (0.729 ± 0.167) and in pancreatitis tissues (0.923 ± 0.184). NQO1 activity was also higher in smokers (2.43 ± 0.61 nmol/min per mg protein) compared to nonsmokers (0.44 ± 0.05 nmol/min per mg protein; p < 0.05). No differences were found in genotype distribution and frequencies of the variant alleles between normal and cancer tissues in this relatively small sample pool. Seventy-five percent of the normal pancreatic tissues showed 609(C/C) and 25% 609(C/T). In pancreatic adenocarcinomas the frequency distribution was 65% C/C, 30% C/T and 5% T/T. The increased expression in noncancer pancreatic tissue from smokers and the fact that smoking is a moderate risk factor for pancreatic cancer suggest that NQO1 expression may be a good candidate as a biomarker for pancreatic cancer, especially in risk groups such as smokers.     

Acquired defects in CFTR-dependent β-adrenergic sweat secretion in chronic obstructive pulmonary disease

Rationale

Smoking-induced chronic obstructive pulmonary disease (COPD) is associated with acquired systemic cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction. Recently, sweat evaporimetry has been shown to efficiently measure β-adrenergic sweat rate and specifically quantify CFTR function in the secretory coil of the sweat gland.

Objectives

To evaluate the presence and severity of systemic CFTR dysfunction in smoking-related lung disease using sweat evaporimetry to determine CFTR-dependent sweat rate.

Methods

We recruited a cohort of patients consisting of healthy never smokers (N = 18), healthy smokers (12), COPD smokers (25), and COPD former smokers (12) and measured β-adrenergic sweat secretion rate with evaporative water loss, sweat chloride, and clinical data (spirometry and symptom questionnaires).

Measurements and main results

β-adrenergic sweat rate was reduced in COPD smokers (41.9 ± 3.4, P < 0.05, ± SEM) and COPD former smokers (39.0 ± 5.4, P < 0.05) compared to healthy controls (53.6 ± 3.4). Similarly, sweat chloride was significantly greater in COPD smokers (32.8 ± 3.3, P < 0.01) and COPD former smokers (37.8 ± 6.0, P < 0.01) vs. healthy controls (19.1 ± 2.5). Univariate analysis revealed a significant association between β-adrenergic sweat rate and female gender (β = 0.26), age (−0.28), FEV₁% (0.35), dyspnea (−0.3), and history of smoking (−0.27; each P < 0.05). Stepwise multivariate regression included gender (0.39) and COPD (−0.43) in the final model (R² = 0.266, P < 0.0001).

Conclusions

β-adrenergic sweat rate was significantly reduced in COPD patients, regardless of smoking status, reflecting acquired CFTR dysfunction and abnormal gland secretion in the skin that can persist despite smoking cessation. β-adrenergic sweat rate and sweat chloride are associated with COPD severity and clinical symptoms, supporting the hypothesis that CFTR decrements have a causative role in COPD pathogenesis.     

Radon progeny activity in sweat following radon exposure in a warm and humid environment

The objective was to evaluate whether activity of radon progenies can be detected in sweat following speleotherapeutic radon exposure (40 kBq/m³) in a warm (38°C) and humid (relative humidity >70%) environment. A group of 11 male patients with spondyloarthropathy (n=6) or non-inflammatory rheumatic diseases (n=5) underwent a 1-h treatment in the gallery of the Gasteiner Heilstollen, and 20 min after leaving the treatment area radon progeny activity was measured in sweat by utilizing a special filter set. The results suggest that radon is discharged with sweat, causing a significant activity of radon and radon progenies on the skin. This finding may be important from a clinical point of view, since specialists experienced in radon therapy have repeatedly emphasized the importance of the degree of radioactivity on the skin for the effectiveness of treatment. It has even been claimed that the skin is the major target for radon therapy, possibly because of the influence on Langerhans’ cell function. Received: 23 August 1999 / Accepted: 1 February 2000     

Regional differences in sweat rate response of steers to short-term heat stress

Six Angus steers (319 ± 8.5 kg) were assigned to one of two groups (hot or cold exposure) of three steers each, and placed into two environmental chambers initially maintained at 16.5–18.8°C air temperature (T _a). Cold chamber T _a was lowered to 8.4°C, while T _a within the hot chamber was increased to 32.7°C over a 24-h time period. Measurements included respiration rate, and air and body (rectal and skin) temperatures. Skin temperature was measured at shoulder and rump locations, with determination of sweat rate using a calibrated moisture sensor. Rectal temperature did not change in cold or hot chambers. However, respiration rate nearly doubled in the heat (P < 0.05), increasing when T _a was above 24°C. Skin temperatures at the two locations were highly correlated (P < 0.05) with each other and with T _a. In contrast, sweat rate showed differences at rump and shoulder sites. Sweat rate of the rump exhibited only a small increase with T _a. However, sweat rate at the shoulder increased more than four-fold with increasing T _a. Increased sweat rate in this region is supported by an earlier report of a higher density of sweat glands in the shoulder compared to rump regions. Sweat rate was correlated with several thermal measurements to determine the best predictor. Fourth-order polynomial expressions of short-term rectal and skin temperature responses to hot and cold exposures produced r values of 0.60, 0.84, and 0.98, respectively. These results suggest that thermal inputs other than just rectal or skin temperature drive the sweat response in cattle.     

Detection of cocaine and cocaethylene in sweat by solid-phase microextraction and gas chromatography/mass spectrometry

In the present work, a semi-quantitative method was developed to detect simultaneously cocaine (COC) and cocaethylene (CE) (transesterification product of the coingestion of COC with ethanol) in sweat. Sweat samples were collected by means of a non-occlusive sweat patch device supplied by PharmChek. The method was based on the dissolution of COC and CE incorporated into the patch, with 0.2 M sodium acetate buffer (pH 5.0) and the extraction of the analytes by solid-phase microextraction (SPME). Gas chromatography/mass spectrometry (GC-MS) was used to detect the analytes in selected ion monitoring mode (SIM). The method showed to be very simple, rapid and sensitive. The limits of detection were 5 ng/ml for COC and CE (12.5 ng/patch). Good inter and intra-assay precision was also observed (coefficient of variation <8%) with the use of deuterated internal standards.     

Intracellular potassium activity and the role of potassium in transepithelial salt transport in the human reabsorptive sweat duct

Summary We have measured the intracellular potassium activity, [K⁺]_i and the mechanisms of transcellular K⁺ transport in reabsorptive sweat duct (RSD) using intracellular ion-sensitive microelectrodes (ISMEs). The mean value of [K⁺]_i in RSD is 79.8±4.1mm (n=39). Under conditions of microperfusion, the [K⁺]_i is above equilibrium across both the basolateral membrane, BLM (5.5 times) and the apical membrane, APM (7.8 times). The Na⁺/K⁺ pump inhibitor ouabain reduced [K⁺]_i towards passive distribution across the BLM. However, the [K⁺]_i is insensitive to the Na⁺/K⁺/2 Cl^– cotransport inhibitor bumetanide in the bath. Cl^– substitution in the lumen had no effect on [K⁺]_i. In contrast, Cl^– substitution in the bath (basolateral side) depolarized BLM from –26.0±2.6 mV to –4.7^*±2.4 mV (n=3;^* indicates significant difference) and decreased [K⁺]_i from 76.0±15.2mm to 57.7^* ±12.7mm (n=3). Removal of K⁺ in the bath decreased [K⁺]_i from 76.3±15.0mm to 32.3^*±7.6mm (n=4) while depolarizing the BLM from –32.5±4.1 mV to –28.3^*±3.0 mV (n=4). Raising the [K⁺] in the bath by 10-fold increased [K⁺]_i from 81.7±9.0mm to 95.0^*±13.5mm and depolarized the BLM from –25.7±2.4 mV to –21.3^*±2.9 mV (n=4). The K⁺ conductance inhibitor, Ba²⁺, in the bath also increased [K⁺]_i from 85.8±6.7mm to 107.0^*±11.5mm (n=4) and depolarized BLM from –25.8±2.2 mV to –17.0^*±3.1 mV (n=4). Amiloride at 10^–6 m increased [K⁺]_i from 77.5±18.8mm to 98.8^*±21.6mm (n=4) and hyperpolarized both the BLM (from –35.5±2.6 mV to –47.8^*±4.3 mV) and the APM (from –27.5±1.4 mV to –46.0^* ±3.5 mV,n=4). However, amiloride at 10^–4 m decreased [K⁺]_i from 64.5±0.9mm to 36.0^*±9.9mm and hyperpolarized both the BLM (from –24.7±1.4 mV to –43.5^*±4.2 mV) and APM (from –18.3±0.9 mV to –43.5^*±4.2 mV,n=6). In contrast to the observations at the BLM, substitution of K⁺ or application of Ba²⁺ in the lumen had no effect on the [K⁺]_i or the electrical properties of RSD, indicating the absence of a K⁺ conductance in the APM. Our results indicate that (i) [K⁺]_i is above equilibrium due to the Na⁺/K⁺ pump; (ii) only the BLM has a K⁺ conductance; (iii) [K⁺]_i is subject to modulation by transport status; (iv) K⁺ is probably not involved in carrier-mediated ion transport across the cell membranes; and (v) the RSD does not secrete K⁺ into the lumen.     

Application of Plasma Levels of Olanzapine and N-Desmethyl-Olanzapine to Monitor Clinical Efficacy in Patients with Schizophrenia

Background

This therapeutic drug monitoring (TDM) study aimed to determine the role of olanzapine (OLZ) and N-desmethyl-OLZ (DMO) levels in the therapeutic efficacy of OLZ in patients with schizophrenia.

Method

Plasma concentrations of OLZ (C_OLZ) and DMO (C_DMO) in schizophrenic patients 12 hours post-dose were assessed. The correlations of C_OLZ and C_DMO with the various scores of the Positive and Negative Syndrome Scale (PANSS) were evaluated. A receiver operating characteristic curve (ROC) was utilized to identify the threshold C_OLZ and C_OLZ/C_DMO ratio for maintenance of satisfactory efficacy.

Results

A total of 151 samples from patients with schizophrenia were analyzed for individual C_OLZ and C_DMO levels. The mean C_OLZ and C_DMO levels were 37.0 ± 25.6 and 6.9 ± 4.7 ng/mL, respectively, and C_OLZ was ~50% higher in female or nonsmokers (p<0.01). In all patients, the daily dose of OLZ was positively correlated with C_OLZ and C_DMO. Linear relationships between C_OLZ and OLZ dose were observed in both nonsmokers and smokers (r_s = 0.306, 0.426, p<0.01), although C_DMO was only correlated with OLZ dose in smokers (r_s = 0.485, p<0.01) and not nonsmokers. In all patients, C_OLZ was marginally negatively correlated with the total PANSS score. The total PANSS score was significantly negatively correlated with the C_OLZ/C_DMO ratio (p<0.005), except in smokers. The ROC analysis identified a C_OLZ/C_DMO ratio ≥2.99 or C_OLZ ≥22.77 ng/mL as a predictor of maintenance of an at least mildly ill status (PANSS score ≤58) of schizophrenia in all patients.

Conclusions

A significantly negative correlation between the steady-state C_OLZ/C_DMO ratio and total PANSS score was observed in Taiwanese schizophrenic patients. TDM of both OLZ and DMO levels could assist clinical practice when individualizing OLZ dosage adjustments for patients with schizophrenia.     

Mu Opioid Receptor Binding Correlates with Nicotine Dependence and Reward in Smokers

The rewarding effects of nicotine are associated with activation of nicotine receptors. However, there is increasing evidence that the endogenous opioid system is involved in nicotine''s rewarding effects. We employed PET imaging with [¹¹C]carfentanil to test the hypotheses that acute cigarette smoking increases release of endogenous opioids in the human brain and that smokers have an upregulation of mu opioid receptors (MORs) when compared to nonsmokers. We found no significant changes in binding potential (BP_ND) of [¹¹C]carfentanil between the placebo and the active cigarette sessions, nor did we observe differences in MOR binding between smokers and nonsmokers. Interestingly, we showed that in smokers MOR availability in bilateral superior temporal cortices during the placebo condition was negatively correlated with scores on the Fagerström Test for Nicotine Dependence (FTND). Also in smokers, smoking-induced decreases in [¹¹C]carfentanil binding in frontal cortical regions were associated with self-reports of cigarette liking and wanting. Although we did not show differences between smokers and nonsmokers, the negative correlation with FTND corroborates the role of MORs in superior temporal cortices in nicotine addiction and provides preliminary evidence of a role of endogenous opioid signaling in frontal cortex in nicotine reward.     

The effect of sodium balance on sweat sodium secretion and plasma aldosterone concentration

The effect of manipulating sodium intake upon sweat sodium secretion was investigated during heat acclimation. Twenty-five male subjects were confined to an environmental chamber at a temperature of 25°C for 3 days, and then acclimated to heat by a further 5 days at 40°C. The subjects' daily sodium intake was controlled throughout as follows: high (HNa), 348.4 (0.8) mmol · day⁻¹, n = 7; moderate (MNa), 174.1 (0.6) mmol · day⁻¹, n = 9; or low (LNa), 66.3 mmol · day⁻¹, n = 9. Sodium losses were estimated from urinary, faecal and sweat collections using a whole-body washdown method. Plasma aldosterone concentration was also measured from venous blood sampled each morning. Measurements of body temperature and heart rate during the heat exposure phase indicated a degree of heat acclimation. During this heat phase there was a reduction (P < 0.01) in sweat sodium secretion for all three conditions which was greatest for the LNa condition, although this finding was not significant (P < 0.1). In the LNa condition, plasma aldosterone concentration increased (P < 0.05) prior to heat exposure, and the secretion of aldosterone was potentiated (P < 0.01) during the heat exposure in comparison with the MNa condition. In contrast, the HNa diet produced a fall (P < 0.05) in plasma aldosterone concentration prior to heat exposure and an attenuation of aldosterone secretion thereafter. These findings are inconsistent with the hypothesis that retention of sweat sodium is dependent upon a net body sodium deficit, but demonstrate that aldosterone secretion is potentiated under such conditions. Accepted: 22 May 1988     

Smoking Is Associated with Shortened Airway Cilia

Background

Whereas cilia damage and reduced cilia beat frequency have been implicated as causative of reduced mucociliary clearance in smokers, theoretically mucociliary clearance could also be affected by cilia length. Based on models of mucociliary clearance predicting that cilia length must exceed the 6–7 µm airway surface fluid depth to generate force in the mucus layer, we hypothesized that cilia height may be decreased in airway epithelium of normal smokers compared to nonsmokers.

Methodology/Principal Findings

Cilia length in normal nonsmokers and smokers was evaluated in aldehyde-fixed, paraffin-embedded endobronchial biopsies, and air-dried and hydrated samples were brushed from human airway epithelium via fiberoptic bronchoscopy. In 28 endobronchial biopsies, healthy smoker cilia length was reduced by 15% compared to nonsmokers (p<0.05). In 39 air-dried samples of airway epithelial cells, smoker cilia length was reduced by 13% compared to nonsmokers (p<0.0001). Analysis of the length of individual, detached cilia in 27 samples showed that smoker cilia length was reduced by 9% compared to nonsmokers (p<0.05). Finally, in 16 fully hydrated, unfixed samples, smoker cilia length was reduced 7% compared to nonsmokers (p<0.05). Using genome-wide analysis of airway epithelial gene expression we identified 6 cilia-related genes whose expression levels were significantly reduced in healthy smokers compared to healthy nonsmokers.

Conclusions/Significance

Models predict that a reduction in cilia length would reduce mucociliary clearance, suggesting that smoking-associated shorter airway epithelial cilia play a significant role in the pathogenesis of smoking-induced lung disease.     

Smoking-related DNA adducts in anal epithelium

Several studies have identified tobacco smoking as a risk factor for anal cancer in both women and men. Samples of anal epithelium from haemorrhoidectomy specimens from current smokers (n=20) and age-matched life-long non-smokers (n=16) were analysed for DNA adducts by the nuclease P₁ digestion enhancement procedure of ³²P-postlabelling analysis. The study included 14 men and 22 women. Both qualitative and quantitative differences in the adduct profiles were observed between the smokers and non-smokers. The mean adduct level was significantly higher in the smokers than in the non-smokers (1.88±0.71 (S.D.) versus 1.36±0.60 adducts per 10⁸ nucleotides, P=0.02, two-tailed unpaired t-test with Welch’s correction); furthermore, the adduct pattern seen in two-dimensional chromatograms revealed the smoking-related diagonal radioactive zone in 17/20 smokers, but not in any of the non-smokers (P<0.00001, Fisher’s exact test). These results indicate that components of tobacco smoke inflict genotoxic damage in the anal epithelium of smokers and provide a plausible mechanism for a causal association between smoking and anal cancer.     

Work in progress: Prostaglandin activity in human eccrine sweat

Thermal sweat collected from 14 healthy volunteers contained prostaglandin activity ranging from 0.2 to 1.5 ng of prostaglandin E₂ equivalents per milliliter (mean ± SE; 0.62 ± 0.43) as measured by a prostaglandin-specific radioimmunoassay. There was no statistically significant difference in the mean activity between males and females. The activity in sweat from two of five patients with atopic dermatitis, in one of four patients with psoriasis, and in one of two patients with hyperhidrosis was significantly ( ) above the mean activity in the sweat of the healthy volunteers. The importance of this prostaglandin activity is uncertain, but prostaglandins may participate in the regulation of sweat electrolytes, as suggested by their influence on electrolyte fluxes in the kidney and gut.     

Nicotine nasal spray with nicotine patch for smoking cessation: randomised trial with six year follow up

ObjectiveTo evaluate the efficacy of using a nicotine patch for 5 months with a nicotine nasal spray for 1 year.DesignPlacebo controlled, double blind trial.SettingReykjavik health centre.Subjects237 smokers aged 22-66 years living in or around Reykjavik.InterventionsNicotine patch for 5 months with nicotine nasal spray for 1 year (n=118) or nicotine patch with placebo spray (n=119). Treatment with patches included 15 mg of nicotine for 3 months, 10 mg for the fourth month, and 5 mg for the fifth month, whereas nicotine in the nasal spray was available for up to 1 year. Both groups received supportive treatment.ResultsThe log rank test for 6 years (χ²=8.5, P=0.004) shows a significant association between abstinence from smoking and type of treatment. Sustained abstinence rates for the patch and nasal spray group and patch only group were 51% v 35% after 6 weeks (P=0.011 (χ²), 95% confidence interval 1.17% to 3.32%), 37% v 25% after 3 months (P=0.045, 1.01% to 3.08%), 31% v 16% after 6 months (P=0.005, 1.27% to 4.50%), 27% v 11% after 12 months (P=0.001, 1.50% to 6.14%), and 16% v 9% after 6 years (P=0.077, 0.93% to 4.72%).ConclusionsShort and long term abstinence rates show that the combination of using a nicotine patch for 5 months with a nicotine nasal spray for 1 year is a more effective method of stopping smoking than using a patch only. The low percentage of participants using the nasal spray at 1 year, and the few relapses during the second year, suggest that it is not cost effective to use a nasal spray for longer than 7 months after stopping a patch.

Key messages

• Combined methods of nicotine replacement therapy have a potential advantage over one method because of high levels of substitution
• Nicotine patches release nicotine slowly, but nicotine nasal spray delivers nicotine more rapidly, enabling the smoker to respond quickly to any smoking urges
• Treatment with a patch and nicotine nasal spray was significantly more effective than patch and placebo from day 15 after stopping smoking
• Using a patch for 5 months with a nicotine nasal spray for 1 year provides a more effective means of stopping smoking than using a patch only
• It is not cost effective to use a nicotine nasal spray for longer than 7 months after stopping a patch

     

TPA Increases Conductance but Decreases Permeability in Neonatal Rat Cardiomyocyte Gap Junction Channels

Short-term (10 min) effects of 100 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), the protein kinase C (PKC) activator, on cardiac macroscopic (g_j) and single channel (γ_j) gap junctional conductances were studied in pairs of neonatal rat cardiomyocytes. Under dual whole-cell (WC) or perforated patch (PP) voltageclamp, g_j increased by 15.5 ± 7.2% (mean ± SD, n = 9) and by 46.3 ± 17.0% (n = 5), respectively. The latter difference is not related to intracellular calcium concentration, because raising the Ca²⁺ concentration in the electrode solution did not change the TPA-induced increase in g_j observed under WC conditions. The inactive phorbol ester, 4α-phorbol 12,13-didecanoate (αPDD), did not affect g_j. Single cardiac gap junction channel events, resolved in the presence of heptanol, indicated two γ_j sizes of 20 and 40-45 pS. Under control conditions, the larger events were most frequently observed. Whereas αPDD did not change this distribution, TPA shifted the γ_j distribution to the lower sizes. Diffusion of Lucifer Yellow (LY) and 6-carboxyfluorescein (6-CF), gap junction permeant tracers, was studied on small clusters of cardiomyocytes. Under control conditions, LY labeled 19.4 ± 7.2 cells (mean ± SD, n = 18) and 6-CF labeled 8.4 ± 2.2 cells (n = 20). Whereas αPDD did not change the extent of dye transfer, TPA restricted the diffusion of LY to 2.8 ± 1.3 cells (n = 11) and of 6-CF to 2.4 ± 1.4 cells (n = 20). This suggests that permeability and single channel conductance of connexin 43 channels are parallely related. Altogether, these results point to the opposite modulation of electrical and metabolic coupling of cardiac cells evoked by TPA.     

Pressure- and parathyroid-hormone-dependent Ca2+ transport in rabbit connecting tubule: Role of the stretch-activated nonselective cation channel

To characterize the Ca²⁺ transport process across the apical membrane of the rabbit connecting tubule (CNT), we examined the effects of luminal pressure on parathyroid hormone (PTH)-dependent apical Ca²⁺ transport in this segment perfused in vitro. An increase of perfusion pressure (0.2 to 1.2 KPa) caused cytoplasmic free Ca²⁺ concentration ([Ca²⁺].) to increase by 42 ± 11 nm in Fura-2 loaded perfused CNT. The response was accentuated when 10 nm PTH was added to the bath (101 ± 30 nm, n = 6). Addition of 0.1 mm chlorphenylthio-cAMP (CPT-cAMP) to the bath also augmented the [Ca²⁺]; response to pressure from 36 ± 16 to 84 ± 26 nm (n = 3). Under steady perfusion pressure at 1.2 KPa, PTH (10 nm) increased [Ca²⁺]; by 31 ± 7 nm (n = 5), whereas it did only slightly by 6 ± 2 nm (n = 12) at 0.2 KPa. The pressure-dependent increase of [Ca²⁺]; was abolished by removing luminal Ca²⁺ (n = 3), and was not affected by 0.1 and 10 m nicardipine (n = 4) in the presence of 10 nm PTH. Cell-attached patch clamp studies on the apical membrane of everted CNT with pipettes filled with either 200 mm CaCl₂ or 140 mm NaCl revealed channel activities with conductances of 42 ± 2 pS (n = 4) or 173 ± 7 pS (n = 5), respectively. An application of negative pressure (–4.9 KPa) to the patch pipette augmented its mean number of open channels (NP ₀ ) from 0.005 ± 0.001 to 0.022 ± 0.005 in the Ca²⁺-filled pipette, and was further accelerated to 0.085 ± 0.014 (n = 3) by 0.1 mm CPT-cAMP. In the Na⁺-filled pipette, similar results were obtained (n = 3), and CPT-cAMP did not activate the stretch-activated channel in the absence of negative pressure (n = 3). These results suggest that a stretch-activated nonselective cation channel exists in the apical membrane of the CNT and that it is activated by PTH in the presence of hydrostatic pressure, allowing entry of Ca²⁺ transport from the apical membrane.We appreciate Ms. Hisayo Hosaka and Ms. Yuki Oyama for their technical assistance and Ms. Keiko Sakai for her secretarial work. This research was supported by grants from the Ministry of Education and Culture of Japan (No. 05670054) and from Yamanouchi Foundation for Research on Metabolic Disorders (1992–1993).     

Apical Heterotrimeric G-proteins Activate CFTR in the Native Sweat Duct

Other than the fact that the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel can be activated by cAMP dependent kinase (PKA), little is known about the signal transduction pathways regulating CFTR. Since G-proteins play a principal role in signal transduction regulating several ion channels [4, 5, 9], we sought to test whether G-proteins control CFTR Cl⁻ conductance (CFTR G _Cl ) in the native sweat duct (SD). We permeabilized the basolateral membrane with α-toxin so as to manipulate cytosolic nucleotides. We activated G-proteins and monitored CFTR G _Cl activity as described earlier [20, 23, 25]. We now show that activating G-proteins with GTP-γ-S (100 μm) also activates CFTR G _Cl in the presence of 5 mm ATP alone (without exogenous cAMP). GTP-γ-S increased CFTR G _Cl by 44 ± 20 mS/cm² (mean ±se; n= 7). GDP (10 mm) inhibited G-protein activation of CFTR G _Cl even in the presence of GTP-γ-S. The heterotrimeric G-protein activator (AlF₄ ⁻) in the cytoplasmic bath activated CFTR G _Cl (increased by 51.5 ± 9.4 mS/cm² in the presence of 5 mm ATP without cAMP, n= 6), the magnitude of which was similar to that induced by GTP-γ-S. Employing immunocytochemical-labeling techniques, we localized Gαs, Gαi, Gαq, and Gβ at the apical membranes of the sweat duct. Further, we showed that the mutant CFTR G _Cl in ducts from cystic fibrosis (CF) subjects could be partially activated by G-proteins. The magnitude of mutant CFTR G _Cl activation by G-proteins was smaller as compared to non-CF ducts but comparable to that induced by cAMP in CF ducts. We conclude that heterotrimeric G-proteins are present in the apical membrane of the native human sweat duct which may help regulate salt absorption by controlling CFTR G _Cl activity. Received: 9 June 2000/Revised: 5 October 2000     

Purification of chemically synthesised dinucleoside(5′,5′) polyphosphates by displacement chromatography

Dinucleoside(5′,5′) polyphosphates (Ap_nA, Ap_nG, Gp_nG, n=3–6) are new group of hormones controlling important biological processes. Because some of the dinucleoside(5′,5′) polyphosphates are commercially not available purification of chemical synthesised dinucleoside(5′,5′) polyphosphates became necessary in order to test their physiological and pharmacological properties. It was the aim of this study to find a method which allows purification of 0.1–0.2 g quantities of dinucleoside polyphosphates by analytical HPLC columns yielding products with impurities lower than 1.0%. Adenosine(5′)-polyphospho-(5′)guanosines were synthesised by mixing the corresponding mononucleotides. The reaction results in a complex mixture of Ap_nA, Ap_nG and Gp_nG (with n=3–6 in all cases). The reaction mixture was concentrated on a preparative C₁₈ reversed-phase column. The concentrate was displaced on a reversed-phase stationary. As a result of displacement chromatography, anion-exchange chromatography in gradient modus yielded baseline separated dinucleoside polyphosphates (homogeneity of the fractions>99%). The identity of the substances were determined by matrix assisted laser desorption ionisation mass spectrometry.