Potassium conductance of smooth muscle cells from rabbit aorta in primary culture

Vascular smooth muscle cells were obtained from rabbit aorta and were studied in primary culture on days 1–7 after seeding with electrophysiological techniques. In impalement experiments a mean membrane potential difference (PD) of –50±0.3 mV (n=387) was obtained with Ringer-type solution in the bath. PD was depolarized by 6±0.3 mV (n=45) and 16±2 mV (n= 5) when the bath K⁺ concentration was increased from the control value of 3.6 mmol/l to 13.6 and 23.6 mmol/l, respectively. Ba²⁺ (0.1–1 mmol/l) depolarized PD. Tetraethylammonium (TEA, 10 mmol/l) depolarized PD only slightly but significantly. Verapamil (0.1 mmol/l) and charybdotoxin (10 nmol/l) had no effect on PD. The conductance properties of these cells were further examined with the patch-clamp technique. K⁺ channels were spontaneously present in cell-attached patches. When the pipette was filled with 145 mmol/l KCl, a mean conductance (g _K) of 209.6±4.6 mV (n=17) was read from the current/voltage curves at a clamp voltage (V _c) of 0 mV. After excision K⁺ channels were found in 129 patches with inside-out and in 50 with outside-out configuration. With KCl on one and NaCl on the other side the mean g _K at a V _c of 0 mV was 134.6±3.9 pS (n=179). The mean permeability was 0.89±0.03×10^–12 cm³/s. With symmetrical KCl solution the mean g _K was 227±6 pS (n=17). The conductance sequence was g _K g _Rb= g _Cs=g _Na=0. TEA blocked dose-dependently only from the outside.(1–10 mmol/l). Lidocaine (5 mmol/l) quinidine (0.01–1 mmol/l) and quinine (0.01–1 mmol/l) blocked from both sides. Charybdotoxin (0.5–5 nmol/l) blocked only from the extracellular side. Ba²⁺ blocked from the cytosolic side and the inhibition was increased by depolarization and reduced by hyperpolarization. At a V _c of 0 mV a half-maximal inhibition (IC₅₀) of 2 mol/l was obtained. Verapamil and diltiazem blocked from both sides, verapamil with an IC₅₀ of 2 mol/l and diltiazem with an IC₅₀ of 10 mol/l. The open probability of this channel was increased by Ca²⁺ on the cytosolic side at activities > 0.1 mol/l. Half-maximal activation occurred at Ca²⁺ activities exceeding 1 mol/l. The present data indicate that the vascular smooth muscle cells of rabbit aorta in primary culture possess a K⁺ conductance. In excised patches only a maxi K⁺ channel was detected. This channel has properties different from the macroscopic K⁺ conductance. Hence, it is likely that the K⁺ conductance of the intact cell is dominated by yet another and thus far not detected K⁺ channel.Supported by DFG Gr 480/10     

Effect of depolarizing and hyperpolarizing agents on the membrane potential difference of primary cultures of rabbit aorta vascular smooth muscle cells

Vascular smooth muscle cells of rabbit aorta were enzymatically dispersed, kept in primary culture, and studied between days 1 and 7 in a bath rinsed with Ringer-like solution at 37°C. The electrical membrane potential difference (PD) was measured with microelectrodes. The mean value of PD was –50±0.4 mV (n=53). Cromakalim (BRL 34915), 1 mol/l and 10 mol/l, hyperpolarized the membrane potential by 9±1 mV (n=11) and 15±1 mV (n=53) respectively. Glibenclamide (10 mol/l) abolished the hyperpolarizing effect of cromakalim (n=6). Simultaneous addition of cromakalim and glibenclamide (both 10 mol/l, n=11) and glibenclamide itself (10 mol/l, n=7) had no effect on PD. In patch-clamp experiments in outside-out-oriented Ca²⁺-sensitive K⁺ channels, cromakalim increased the open probability (P _o) only slightly and only with a cytosolic Ca²⁺ activity of 1 mol/l. In all other series cromakalim had no effect on the P _o of these channels. Forskolin (10 mol/l) hyperpolarized PD by 6±1 mV (n=13). The nucleotides UTP, ATP and ITP (10 mol/l) depolarized PD by 12±1 mV (n=7), 8±1 mV (n=65) and 5±1 mV (n=6) respectively. GTP, [,-methylene]ATP and adenosine had no significant effect. Mn²⁺ (1 mmol/l, n=18), Ni²⁺ (1 mmol/l, n=13), Co²⁺ (1 mmol/l, n=11), Zn²⁺ (1 mmol/l, n=6) and the Ca²⁺-channel blockers verapamil and nifedipine (both 0.1 mmol/l, n=6) did not attenuate the depolarization induced by 10 mol/l ATP. Fetal calf serum (100 ml/l, n=7) depolarized PD by 11±2 mV. This effect was not abolished by nifedipine or by replacing NaCl by choline chloride. The data indicate that PD of vascular smooth muscle cells is depolarized by P₂ agonists and hyperpolarized by the K⁺-channel opener cromakalim. The effect of cromakalim is antagonized by glibenclamide. The effect of cromakalim is probably not mediated by the K⁺ channel identified in excised patches.Supported by DFG Gr 480/10     

Ion conductances of isolated cortical collecting duct cells

The study of ion conductances in the intact cortical collecting duct (CCD) with the patch-clamp method is rather difficult. An optimized method to isolate CCD cells from rat kidneys using an in vivo followed by an in vitro enzyme digestion is described. Individual CCD segments were collected after this digestion and incubated in EGTA-buffered medium. This procedure resulted in single cells or cell clusters. These freshly isolated CCD cells were studied with different modifications of the patch-clamp method. Membrane voltages measured in the cell-attached-nystatin configuration were –74 ±1mV (n=13) and –68±3 mV (n=22) in cells isolated from normal and mineralocorticoid-treated rats respectively. These values and those measured with the nystatin-perforated slow-whole-cell configuration (–79 ±1mV, n=23) are comparable to those measured in principal cells of isolated CCD segments. The cells hyperpolarized after the addition of amiloride and depolarized with the addition of adiuretin to the bath. The amiloride effect was enhanced when cells were isolated from deoxycorticosterone-acetate-treated rats. The cells were strongly depolarized upon elevation of the extracellular K⁺-concentration and did not demonstrate a measurable Cl^– conductance. A large-conductance K⁺ channel (174 pS, n=5, cell-attached, 145 mmol/l K⁺ in the pipette; 140 pS, n=12, cell-free, 3.6 mmol/l K⁺ in the bath) was seen. It had a very low activity on the cell, but a high open probability when excised into a solution with 1 mmol/l Ca²⁺ on the cytosolic side. More often a small-conductance K⁺ channel (36–52 pS, n=19, cell-attached; 30 pS, n=5, cell-free) with a high open probability was found on the cell. These freshly isolated cells seem to be a powerful preparation to study the properties and regulation of ion conductances of rat CCD with several electrophysiological methods. These freshly isolated CCD cells maintain the conductance properties known from principal cells of the intact CCD.     

Handling of allantoin by the rat kidney

Summary Renal excretion of allantoin was measured by tracer techniques. After injection of 2-C14 urate and H3 inulin, clearances of allantoin and inulin were measured and both proximal and distal tubules were micropunctured.In confirmation of earlier results 2-C14 urate injected into an intact animal is very rapidly converted to C14 allantoin: after 15 min more than 90% of urinary tracer is present as allantoin. It was further observed that 1) allantoin clearance is essentially identical with inulin clearance over a wide range of urine flows; 2) no net transport of allantoin occurs in either proximal or distal tubules. Clearly allantoin is handled by the rat kidney like inulin.The total excretion of filtered allantoin unlike that of filtered urate provides an easy and effective mechanism for animals possessing the enzyme uricase to dispose of their purine loads.Partially supported by the Österreichischer Fonds zur Förderung der wissenschaftlichen Forschung.Receiving scholarships from Deutsche Forschungsgemeinschaft.     

pH regulation in HT29 colon carcinoma cells

The pH regulation in HT₂₉ colon carcinoma cells has been investigated using the pH-sensitive fluorescent indicator 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). Under control conditions, intracellular pH (pH_i) was 7.21±0.07 (n=22) in HCO ₃ ^– -containing and 7.21±0.09 (n=12) in HCO ₃ ^– -free solution. HOE-694 (10 mol/l), a potent inhibitor of the Na⁺/H⁺ exchanger, did not affect control pH_i. As a means to acidify cells we used the NH ₄ ⁺ /NH₃ (20 mmol/l) prepulse technique. The mean peak acidification was 0.37±0.07 pH units (n=6). In HCC ₃ ^– -free solutions recovery from acid load was completely blocked by HOE-694 (1 mol/l), whereas in HCO3 ₃ ^– -containing solutions a combination of HOE-694 and 4,4-diisothiocyanatostilbene-2, 2-disulphonate (DIDS, 0.5 mmol/l) was necessary to show the same effect. Recovery from acid load was Na⁺-dependent in HCO ₃ ^– -containing and HCO ₃ ^– -free solutions. Removal of external Cl^– caused a rapid, DIDS-blockable alkalinization of 0.33±0.03 pH units (n=15) and of 0.20±0.006 pH units (n=5), when external Na⁺ was removed together with Cl^–. This alkalinization was faster in HCO ₃ ^– -containing than in HCO ₃ ^– -free solutions. The present observations demonstrate three distinct mechanisms of pH regulation in HT₂₉ cells: (a) a Na⁺/H⁺ exchanger, (b) a HCO ₃ ^– /Cl^– exchanger and (c) a Na⁺-dependent HCC ₃ ^– transporter, probably the Na⁺-HCO ₃ ^– /Cl^– antiporter. Under HCO ₃ ^– — free conditions the Na⁺/H⁺ exchanger fully accounts for recovery from acid load, whereas in HCO ₃ ^– -containing solutions this is accomplished by the Na⁺/H⁺ exchanger and a Na⁺-dependent mechanism, which imports HCO ₃ ^– . Recovery from alkaline load is caused by the HCO ₃ ^– /Cl^– exchanger.This study was supported by DFG Gr 480/10     

Mechanism of NaCl secretion in rectal gland tubules of spiny dogfish (Squalus acanthias)

Rectal gland tubule (RGT) segments of the spiny dogfish (Squalus acanthias) were perfused in vitro. The effects of inhibitors of known mode of action on transepithelial PD (PDte resistance (Rte), the PD across the basolateral membrane (PDbl), the fractional resistance of this membrane (FRbl), and intracellular activities of NA+, Cl-, K+ (apha cell) were examined. Furosemide (5 x 10(-4) mol x 1(-1)) reduced PDte from -12 +/- 0.7 to -2.3 +/- 0.2 mV (n = 63), hyperpolarized PDbl from -71 +/- 1.3 to -79 +/- 0.9 mV (n = 59), FRbl decreased from 0.2 +/- 0.03 to 0.13 +/- 0.01 (n = 21), alpha cell cl- fell from 38 +/- 4 to 11 +/- 2 mmol x 1(-1) (n = 21), alpha cell Na+ fell from 37 +/- 4 to 17 +/- 2 mmol x 1(-1) (n = 12) and alpha cell K+ was constant [113 +/- 14 vs. 117 +/- 15 mmol x 1(-1) (n = 6)]. Furosemide exerted its effects within some 20-40s. Its action was completely reversible. Analysis of the time courses revealed that the furosemide induced initial fall in alpha cell cl- was approximately twice as rapid when compared to that of alpha cell Na+. Ba2+ 0.5 mmol x 1(-1) (bath) reduced PDte from -7.1 +/- 1.2 to -4.1 +/- 0.6 mV (n = 24), increased Rte from 18 +/- 2 to 22 +/- 2.5, omega cm2 (n = 14). PDbl depolarized from -75 +/- 2 to -48 +/- 2 mV (n = 42), FRbl increased from 0.2 +/- 0.02 to 0.34 +/- 0.04 (n = 14) and alpha cell K+ increased from 143 +/-28 to 188 +/- mmol x 1(-1) (n = 4). Ouabain (50 x 10(-6) mol x 1(-1), bath) reduced PDte from -12 +/-2 to -3 +/- 0.5 mV (n = 9), Rte increased from 18 +/- 3 to 21 +/- 3 omega cm2 (n = 5). PDbl depolarized from -67 +/- 4 to -26 + 3 mV (n = 14), FRbl increased from 0.23 +/- 0.04 to 0.45 +/- 0.05 (n = 6), alpha cell K+ fell only slightly from 135 +/- 15 to 112 +/- 30 mmol x 1(-1) (n = 4), but alpha cell cl- increased from 35 +/- 12 to 111 +/- 37 mmol x 1(-1) (n = 3). These effects of ouabain were slow when compared to those exerted by furosemide or Ba2+. The ouabain effects on PDte and PDbl were completely prevented if furosemide was applied first.(ABSTRACT TRUNCATED AT 400 WORDS)     

The “small” conductance chloride channel in the luminal membrane of the rectal gland of the dogfish (Squalus acanthias)

Besides the larger Cl^– channel with a single channel conductance of about 45 pS, a small channel was observed in the luminal membrane of the dogfish rectal gland [9]. In cell excised (inside out) patches with NaCl solution on both sides, the latter channel had a single channel conductance of 11±1 pS (n=21), and its current-voltage relationship was linear in the voltage range+90 to –90 mV. The open state probability increased moderately with negative clamp potentials. Ionic replacement studies revealed a high selectivity of Cl^– over gluconate, sulfate, and iodide, whereas bromide was permeable to some extent. Also the channel is impermeable for Na⁺. The Cl^– channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate did not affect this small conductance Cl^– channel. It can be concluded that the luminal membrane of stimulated rectal gland cells possesses two types of Cl^– channels, which differ markedly in their characteristics.Supported by Deutsche Forschungsgemeinschaft Gr 480/8 and by NSF and NIH grants to the MDIBL     

Mineralocorticoid receptor knockout mice: lessons on Na+ metabolism

The mineralocorticoid receptor (MR) binds aldosterone and glucocorticoids with equal affinity. In aldosterone target tissues, like the epithelial cells of the distal colon and the principal cells of the collecting ducts in the kidney, the MR is protected from glucocorticoids by the action of the enzyme 11beta-hydroxysteroid-dehydrogenase type 2 (11betaOHSD2), allowing aldosterone to specifically activate the receptor. However, in MR-expressing cells, which lack 11betaOHSD2, like the neurons of the limbic system in the brain, MR is mainly activated by glucocorticoids. MR knockout mice die in the second week after birth, showing at day 8 symptoms of pseudohypoaldosteronism with hyponatremia, hyperkalemia, high renal salt wasting, and a strongly activated renin-angiotensin-aldosterone system (RAAS). The activity of the amiloride-sensitive epithelial Na+ channel (ENaC) is strongly reduced in colon and kidney, but there is no down-regulation of the mRNA abundance of the three ENaC subunits. Daily subcutaneous injections of isotonic NaCl solution until weaning and continued oral NaCl supply lead to survival of the MR knockout mice. The NaCl-rescued MR knockout mice display a strongly enhanced fractional renal excretion of Na+, hyperkalemia, and a persistently strongly activated RAAS. There is almost no renal ENaC activity. The renal mRNA abundance of alphaENaC is reduced by 30%, whereas betaENaC and gammaENaC are not altered.     

Virus transmission during orthopedic surgery on patients with COVID-19 – a brief narrative review

Background and purpose — COVID-19 is among the most impactful pandemics that the society has experienced. Orthopedic surgery involves procedures generating droplets and aerosols and there is concern amongst surgeons that otherwise rational precautionary principles are being set aside due to lack of scientific evidence and a shortage of personal protective equipment (PPE). This narrative review attempts to translate relevant knowledge into practical recommendations for healthcare workers involved in orthopedic surgery on patients with known or suspected COVID-19.Patients and methods — We unsystematically searched in PubMed, reference lists, and the WHO’s web page for relevant publications concerning problems associated with the PPE used in perioperative practice when a patient is COVID-19 positive or suspected to be. A specific search for literature regarding COVID-19 was extended to include publications from the SARS epidemic in 2002/3.Results — Transmission of infectious viruses from patient to surgeon during surgery is possible, but does not appear to be a considerable problem in clinical practice. Seal-leakage is a problem with surgical masks. Due to the lack of studies and reports, the possibility of transmission of SARS-CoV-2 from patient to surgeon during droplet- and aerosol-generating procedures is unknown.Interpretation — Surgical masks should be used only in combination with a widely covering visor and when a respirator (N95, FFP2, P3) is not made available. Furthermore, basic measures to reduce shedding of droplets and aerosols during surgery and correct and consistent use of personal protective equipment is important.

Due to the COVID-19 pandemic, elective orthopedic procedures are currently, to a great extent, postponed (CDC 2020, ECDC 2020). However, patients with and without COVID-19, with cancer, infections in bones, joints, and soft tissues, critical ischemia, open and unstable fractures, and other urgent diagnoses will still be in need of orthopedic surgery.It is widely accepted that healthcare workers (HCWs) performing procedures involving the respiratory tract face a high risk of contracting Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). This is reflected in the WHO’s recommendation (WHO 2020b) for the highest standard of personal protective equipment (PPE) in these cases. The risk for HCWs to contract SARS-CoV-2 during orthopedic surgery, or most other surgeries for that matter, has not been studied and is consequently less clear.Orthopedic surgery often involves the use of high-speed saws, power drills, pulsed lavage, suction, and electrocauterization. Shedding of droplets from the wound is reflected in the extensive use of protective eyewear, such as goggles and visor, in everyday practice. Concern amongst surgeons and other HCWs that otherwise rational precautionary principles are being set aside due to lack of scientific evidence and a shortage of PPE is obvious on social media platforms and amongst colleagues.This short review is an attempt to translate relevant knowledge into practical recommendations for HCWs involved in orthopedic surgery on patients with known or suspected COVID-19.Transmission of SARS-CoV-2Virus shedding through droplets that rapidly fall to the ground requires a different PPE approach to prevent transmission than infectious aerosols that can remain airborne for a longer period. According to the WHO, the transmission of SARS-CoV-2 mainly occurs through droplets (WHO 2020a). The organization concludes that transfer through aerosols is unlikely except during specific aerosol-generating procedures. As a response, Nature problematized existing controversies regarding possible air-transmission in a news story, shedding light on the complexity of the subject and that several uncertainties cannot be clarified in a long time (Anon 2020).SARS-CoV-2 RNA (vRNA) has been found in aerosols (Ong et al. 2020, Santarpia et al. 2020). However, it is not clear whether aerosols contain infectious SARS-CoV-2, or enough viable virus to transmit the disease. We are not aware of any studies that have investigated aerosols produced during surgery on patients with SARS-CoV-2 viremia or disseminated disease.Presence of SARS-CoV-2 in the musculoskeletal systemSARS-CoV-2 was first identified in December 2019 and, consequently, is not fully understood. Like SARS-CoV, the coronavirus causing the 2002/3 SARS epidemic, SARS-CoV-2 binds to ACE2 receptors on human cells (Shang et al. 2020). ACE2 receptors are present on cells in the lungs and small intestines, but also on cells in a variety of other tissues, including veins, arteries, and skeletal muscle, throughout the body (Hamming et al. 2004, Riquelme et al. 2014).Most current tests used to confirm the presence of SARS-CoV-2 use PCR technology to detect vRNA. A vRNA test will return positive with viable virus, but also with non-viable virus and virus debris. Only a viable virus can infect new individuals. To our knowledge infectious virus has been found only in respiratory tract tissue and in 2 fecal samples from 8 patients (Wang et al. 2020, Wolfel et al. 2020). We are not aware of any published studies that have aimed to find viable SARS-CoV-2 in blood, bone, bone marrow, or skeletal muscle in COVID-19 patients. An in vitro study showed replication of SARS-CoV-2 within blood-vessel organoids (Monteil et al. 2020).Several studies have identified vRNA in blood and serum (Shang et al. 2020, Wang et al. 2020, Young et al. 2020). vRNA has been found in both the severely ill and in patients with mild symptoms. Amongst 15 patients with multiple site samples, vRNA in blood was detected in 6 patients with negative swabs from the upper respiratory system (Zhang et al. 2020).An autopsy study including 8 deceased patients from the SARS epidemic in 2002/3 showed widespread virus dissemination in immune cells of the blood, spleen, and lymph nodes and in cells of the respiratory tract, renal tubules, intestines, and brain (Gu et al. 2005). Virus was not found within skeletal muscle cells.Aerosol formation during orthopedic surgeryHigh-speed saws, power-drills, pulsed lavage, suction, and electrocauterization are all droplet- and aerosol-generating procedures. Infected fluids, such as blood and irrigation fluid, can aerosolize during surgery and shed bacteria and viruses and have the potential to transmit disease (Heinsohn and Jewett 1993).During experimental set-ups in vivo, infectious HIV-1 particles have been found in aerosols produced using an oscillating saw on a known infected individual (Johnson and Robinson 1991) and aerosols formed in laser fume transmitted disease in a bovine Papillomavirus model (Garden et al. 2002). Both the use of a high-speed cutter and pulsed lavage showed shedding of Staphylococcus aureus several meters from the operating field. The shedding was reduced, but not eliminated, when a drape was used as an overlying protective barrier (Nogler et al. 2001, Putzer et al. 2017). Literature is sparse, and we could not find evidence of disease transmission from patient to surgeon through aerosolized virus-infected fluids from orthopedic-like procedures in clinical practice.Surgical masks and particulate respirators (N95, FFP2/P3)Originally, surgical masks were made to protect the patient from infectious pathogens in HCWs. Respirators, the somewhat confusing technical term for face masks with the standards N95, FFP2/P3, were designed to protect the user from airborne particles.The WHO’s recommendations regarding PPE do not discuss aerosol-generating surgical procedures on infected patients (WHO 2020b). A review from the Norwegian Institute of Public Health (2020) concludes that evidence regarding the risk of aerosol transmission through aerosol-generating procedures, other than those directly or indirectly affecting the airways, is low. Respirators (N95, FFP2/P3), are consequently not recommended for open surgeries elsewhere in the body (WHO 2020b, FHI 2020).A randomized controlled clinical trial including 446 nurses concluded non-inferiority of surgical masks when compared with N95 respirators in preventing transmission of influenza and other respiratory viruses (coronavirus included) from patients to HCWs (Loeb et al. 2009). This finding from clinical practice has been supported by 3 later meta-analyses including approximately 9,000 subjects (Smith et al. 2016, Bartoszko et al. 2020, Long et al. 2020). All the included studies were performed in non-aerosol-generating settings. N95 respirators were found to be superior to surgical masks under laboratory settings regarding filter penetration and face-seal leakage (Smith et al. 2016). Experience from the SARS epidemic stresses the importance of correct and consistent use of PPE and that this might be just as important as type of airway protection to prevent nosocomial disease transmission (Seto et al. 2003, Loeb et al. 2004). It must still be emphasized that data from the SARS outbreak in Toronto showed a trend in favor of N95 respirators over surgical masks for HCWs involved in respiratory tract procedures. The difference did not reach statistical significance, but the number of nurses included was low for this sub-analysis (n = 20, 3 infected) (Loeb et al. 2004).Can virus transmission occur during orthopedic surgery on patients with Covid-19?COVID-19 is a new, harmful and rapidly spreading disease that first occurred less than 4 months ago, i.e., in December 2019. The knowledge regarding the potential of SARS-CoV-2, and the previous SARS-CoV, to spread via droplets and aerosols produced during surgery is very sparse.Some patients with both mild and severe COVID-19 have vRNA in their blood indicative of viremia. Infectious disease transmission through both droplets and aerosols produced during orthopedic surgery is possible. In the case of SARS-CoV-2, the risk naturally depends on the virus’s capability of transmission through tissues other than respiratory tract tissues and feces. Results from possible investigations of such a capability have not been published at the time of writing (April 2020).