Muscarinic modulation of calcium dependent plateau potentials in rat neostriatal neurons

Intracellular recording from neostriatal neurons in rat brain slices revealed effects of the acetylcholine (ACh) agonist carbachol (Cch, 1–10 mol/l), of the anticholinesterase physiostigmine (10 mol/l) and of the muscarinic antagonist atropine (10 mol/l) on plateau potentials elicited in the presence of K-blockers were Cadependent, elicited in the presence of K-blockers were Cadependent, since they persisted in Na-free solution, were resistant to tetrodotoxin (TTX, 3 mol/l) and blocked by Cd (0.1–0.5 mmol/l). Cch reduced the duration of the plateau potentials and made them more susceptible to fatigue. These effects were antagonized by atropine (1–10 mol/l), but not by Ba (100–200 mol/l) or 4-aminopyridine (4-AP, 0.5 mmol/l). Physostigmine (10 mol/l) had the same atropine-sensitive effects as Cch on the plateau potential. Atropine (10 mol/l), by itself, prolonged the duration of the plateau potential. High concentrations (100 mol/l) of Cch did not further reduce the duration of the plateau potential, instead, the duration re-increased with prolonged exposure. The re-increase of the plateau-spike duration was later masked by bursting activity. The opposing effects of low and high concentrations of Cch on the plateau potential duration corresponded to effects of this drug on intrastriatally evoked EPSPs in that low concentrations of Cch reduced the EPSP amplitude, but high concentrations re-increased it after a transient decrease. It is concluded that the muscarinic effect of Ach in the neostriatum is to modulate Ca-influx and that this effect is exerted in a tonic manner. On leave from absence from: Clinica Neurologica II, Universita di Roma. Tor Vergata, Roma, Italy.     

Regulation of potassium conductance by prostaglandins in cultured renal epitheloid (Madin-Darby canine kidney) cells

Madin-Darby canine kidney (MDCK) cells form arachidonic acid metabolites following stimulation of several hormones known to modify the ion conductances at the plasma membrane. The present study has been performed to elucidate the influence of arachidonic acid on the electrical properties of subconfluent MDCK cells. As a result, arachidonic acid (1 or 10 mol/l) leads to a transient hyperpolarization of the cell membrane, followed by a transient depolarization and a second, sustained hyperpolarization. The effects are inhibited by cycloxygenase inhibitor indomethacin (1 mol/l). The initial transient hyperpolarization is mimicked by prostaglandin E₂ (PGE₂, 0.1 mol/l), the sustained hyperpolarization by both PGE₂ (0.1 mol/l) and PGF₂ (0.1 mol/l). The transient hyperpolarization is paralleled by an increase of potassium selectivity and a decrease of cell membrane resistance and is thus the result of increased potassium conductance. The transient depolarization is paralleled by an increase of chloride selectivity, reflecting an increase of chloride conductance. The sustained hyperpolarization is paralleled by an increase of cell membrane resistance, and increase of potassium selectivity and a decrease of chloride selectivity, and is thus the result of decreasing chloride conductance. The observations reveal a role of prostaglandins in the regulation of ion conductances in MDCK cells, which could well participate in the transport regulation by hormones.     

Modulation of lymphocyte activating factor activity (interleukin 1 like activity) and acute phase proteins in pertussis-induced air pouch inflammation by muramyl dipeptide

We have studied the effect of the macrophage activator, muramyl dipeptide (MDP) on immune inflammation induced in the rat six day subcutaneous air pouch. Treated animals received either 100 g or 200 g MDP at the time of challenge and twenty four hours before exudate harvest. Using the thymocyte co-mitogenic assay for lymphocyte activating factor (LAF), 100 g MDP enhanced LAFactivity whereas 200 g caused inhibition. Increased dilution of 200 g exudate in this assay removed this inhibition. Similarly, at the lower dose, MDP caused enhanced production of the acute phase protein alpha 1 glycoprotein, whereas the higher dose had no effect. The present study suggests that macrophage activity can be manipulatedin vivo to produce LAF and naturally occurring inhibitors of LAF. These studies indicate that the stimulation of LAF inhibitors by MDP may be a potential theerapeutic action.     

A simple method for measurement of inulin in nanoliter samples using glass capillaries

Summary A simple method using glass capillaries instead of microcuvettes for measurement of inulin in nanoliter samples is given. Inulin was determined with anthron reagent (5 or 10 nl samples +3 l anthron reagent). Glass capillary tubes (o.d.=1 mm, i.d.=0.68 mm, length=150 mm) in which the chemical reaction took place during incubation at 56°C were directly introduced into the optical system of a Zeiss spectrophotometer PMQ II with sphere attachment and objective.Extinction was measured vertically to the axis of the capillary. The changes of extinction of 20 different capillaries with the blank at different positions was only 1.13×10^–3. The exactness of measurement in the concentration range of 100 200 400 750 1500 3000 mg-% inulin was for 5nl/3 l: 19.8 11.0 6.7 4.7 3.0 2.2%. 10nl/3 l: 13.0 8.4 5.1 3.9%.This method of measurement may also be applicable for other colorimetric reactions with nanoliter samples.This work was supported by Fonds zur Förderung der wissenschaftlichen Forschung.     

Expression of surface membrane IgG on pokeweed mitogen-reactive anti-tetanus toxoid antibody-producing cells

Anti-tetanus toxoid antibody-producing cells, differentially expressing surface membrane IgM, were analyzed for the additional expression of surface membrane IgG. ⁺ and ^– cells were rosetted with anti--ox red blood cells and separated by density centrifugation into fractions enriched or depleted or ⁺ cells. These B-cell subsets were assayed for the production of IgM and IgG anti-tetanus toxoid antibody and total IgM and IgG. The results indicated that the majority of anti-tetanus toxoid antibody synthesis in the ^– fraction was by ⁺ cells. In the ⁺ fraction, however, both IgM and IgG anti-tetanus toxoid antibody production was detected in the ^+– and ⁺⁺ fraction. The inclusion of isotype-specific antisera during the first 2 days of culture further established that was expressed on the surface of the majority of the precursors for IgG anti-tetanus antibody productionin vitro. Studies performed to determine the culture requirements of ^– and ⁺ cells revealed that production of IgG anti-tetanus toxoid antibody by both cell subsets was dependent on T cells and pokeweed mitogen. However, some ^– cells could produce IgG in the presence of T cells alone.     

Effect of epidermal growth factor on non-steroidal anti-inflammatory drug-induced intestinal damage

Epidermal growth factor (EGF, 10 g/kg po, ip, or sc, BID, and 20 g/kg iv) had no protective activity in the indomethacin-induced intestinal lesion model (6 h model). In the ethanol-induced gastric lesion model, EGF (10 g/kg sc) reduced lesions by 52% and reduced gastric acid secretion by 68% (5 g/kg iv). In the 24 h indomethacin-induced intestinal lesion model, pretreatment with EGF (10 g/kg sc, BID; 1 day before and during indomethacin treatment) had no beneficial effects. Therefore, EGF had no protective effects against non-steroidal antiinflammatory drug (NSAID)-induced intestinal lesions at doses that protect against the necrotizing action of ethanol and that inhibit gastric acid secretion in the rat.     

Lectins modulate prostaglandin E2 production by rat peritoneal macrophages

The effect of Aloctin A (Alo A), a lectin having anti-inflammatory activities, on prostaglandin (PG) E₂ production by activated rat peritoneal macrophages was compared with that of concanavalin A (Con A), wheat germ agglutinin (WGA), plsum sativum agglutinin (PSA) and soybean agglutinin (SBA). Alo A, WGA, Con A and PSA at 10 g per ml inhibited PG E₂ production. But SBA, even at a dose of 1 g per ml, stimulated PG E₂ production. The inhibition by Alo A treatment of the release of radioactivity from (³H)arachidonic acid-labeled macrophages and the stimulation of this release by SBA treatment were observed. The uptake of (⁵¹Cr)-labeled sheep red blood cells by the macrophage was inhibited by Alo A, Con A, and PSA, all at 10 g per ml and SBA at 1 g per ml, however, WGA at 10 g per ml stimulated the uptake of the sheep red blood cells. The mechanism of the anti-inflammatory properties of Alo A was discussed.To whom correspondence should be addressed.     

Effects of vector cutting on its recombination with the chromosomal immunoglobulin gene in hybridoma cells

We have analyzed the effects of linearizing vector DNA on the frequency and pathway of its recombination with the homologous chromosomal gene. The pSV2neo vector bearing a 4.3-kb fragment encoding the mouse immunoglobulin heavy chain constant (C) region was cut either at sites within the C segment or outside C and then transferred to hybridoma cells bearing a mutant gene. The frequency of recombinant cells producing normal was then measured. For most cut sites, whether in regions of homology or of nonhomology, linearization of the transferred DNA enhanced the recombination frequency between the vector and chromosomal genes. When the vector was either uncut or cut at SacI in the region of homology, G418-resistant m⁺ recombinants were found to have integrated the vector by a single reciprocal homologous crossover; the enzyme site (SacI) used for cutting was present in the recombinants. By contrast, when the vector had been linearized at Pvul or SfiI in the region of nonhomology, vector integration involved nonhomologous crossovers, either between transferred DNA molecules or between transferred and chromosomal DNA, and the vector cut sites were absent in these recombinants. Some recombinants were found to have an unaltered as well as recombinant gene, suggesting that the nonhomologous recombination process might have involved sister chromatids.     

Control of pulmonary vascular smooth muscle tone by sarcoplasmic reticulum ca2+ pump blockers: thapsigargin and cyclopiazonic acid

The effect of thapsigargin (TG) and cyclopiazonic acid (CPA) on the mechanical activity of the rat pulmonary artery were investigated. In chemically (-escin)-skinned arterial strips, application of TG (0.1–1 M) or CPA (0.5–10 M) prior and throughout the loading procedure of the internal Ca²⁺ stores (0.3 M free Ca²⁺ ions for 8–10 min) concentration dependently inhibited the subsequent contractile response induced by noradrenaline (NA, 10 M) or caffeine (25 mM). In intact strips repeatedly incubated in a Ca²⁺-containing solution (2.5 mM for 10 min), followed by incubation in a Ca²⁺-free solution 12 min before NA-stimulation, TG and CPA not only inhibited the NA-induced contraction but also increased the tension which appeared during the exposure time to Ca²⁺. The two phenomena developed with similar time courses. The increase in tension during the readmission of Ca²⁺ ions was not antagonized by verapamil (10 M) or nifedipine (1 M) but was blocked by La³⁺ (50 M) and Co²⁺ (1 mM) ions. The amplitude of the verapamil-insensitive TG (or CPA)-induced contraction was dependent on the external [Ca²⁺] [0.1–10 mM, concentration for half maximal effect (EC₅₀) =0.85 mM], not modified by the reduction of the external [Na⁺] (from 130 to 10 mM) and decreased by depolarization of the strip using K⁺-rich (30–120 mM) solutions. Under the latter condition, 38±9 and 83±4% reduction (n=5) was observed in the presence of 60 and 120 mM K⁺ respectively. This contraction was also concentration dependently inhibited by the tyrosine kinase inhibitors genistein (0.5–50 M) and tyrphostin (2–50 M). Sr²⁺ ions, which contracted both depolarized intact and skinned strips, failed to replace Ca²⁺ ions in the verapamil-insensitive contraction induced by TG or CPA (n=4). Finally, TG (1 M) and CPA (10 M) did not modify the pCa tension relationship in skinned strips (n=5). These results show that the main action of TG and CPA in rat pulmonary artery is to prevent the refilling of the internal Ca²⁺ store. TG and CPA also seem to facilitate a Ca²⁺ influx through a specific verapamil-insensitive pathway. The biophysical and molecular characteristics of this pathway remain to be elucitated, although it appears to involve a tyrosine kinase activity.     

Morphological studies on equine arteritis virus

Summary Electron microscopy of purified preparations of equine arteritis virus (EAV) revealed enveloped, spherical particles with an average diameter of 55 m. The envelope was found to carry tiny projections on the surface, 3 to 5 m in length. No detailed structure of an internal component could be seen.In sections of EAV infected BHK cells 24 hours after inoculation, viral particles were shown to bud from the cytoplasmic matrix into cisternae of the endoplasmic reticulum, the process starting in the vicinity of the Golgi apparatus. The mature particles had an average diameter of 50 m, with an inner core measuring 25m.This study was supported by grant no. B70-16X-744-05-6068-19195 from the Swedish Medical Research Council.     

Arachidonic acid inhibits hormone-stimulated cAMP accumulation in the medullary thick ascending limb of the rat kidney by a mechanism sensitive to pertussis toxin

The possible regulation of adenosine 3,5-cyclic monophosphate (cAMP) accumulation by arachidonic acid (AA) was studied in segments, microdissected from the rat kidney, which are sensitive to arginine vasopressin (AVP). In the presence of 5 M indomethacin, the addition of 5 M AA did not impair AVP-dependent cAMP accumulation (measured during 4 min at 35° C) in the cortical or outer medullary collecting tubule, but decreased this response in the thick ascending limb with an inhibition much more pronounced in the medullary portion (MTAL) than in the cortical portion. In MTAL, the response to 10 nM AVP was inhibited by 34.4±9.6% (SEM) and 65.8±5.4% with 1 M and 5 M AA, respectively, N=5 experiments. AVP-, glucagon- and calcitonin-sensitive cAMP levels in MTAL were inhibited by 5 M AA to a similar extent. AA-induced inhibition was unaffected by the presence of inhibitors of AA metabolism: (1) either 10 M indomethacin or 50 M ibuprofen added to all media; (2) a 10-min pre-incubation and a 4-min incubation of MTAL samples with 10 M eicosa-5,8,11,14-tetrayonic acid, (3) a 1-h preincubation with either 30 M SKF-525A, 20 M ketoconazole, or 20 M nordihydroguariaretic acid. In contrast to AA, 11 other saturated or unsaturated fatty acids had no inhibitory effect on the AVP-dependent cAMP level. In fura-2-loaded MTAL samples, AA induced a slow increase of the intracellular calcium concentration ([Ca²⁺]_i) which reached 21.0±3.8 nM and 92.9 ±21.4 nM over basal values (n=11) at 2 min and 4 min, respectively, after the beginning of the superfusion of 5 M AA. AA-induced inhibition of AVP-dependent cAMP accumulation was due neither to the increase in [Ca²⁺]_i elicited by AA, nor to an activation of protein kinase C because this inhibition: (1) was not blocked when MTAL samples were incubated either in zero Ca²⁺ medium, or in the presence of 1,2-bis(2-aminophenoxy)ethane-N, N, N N-tetraacetic acid (BAPTA) to chelate [Ca²⁺ _i, and (2) it was not reproduced by a pre-treatment of MTAL segments with a phorbol ester. Pre-incubation of MTAL (6 h at 35° C) with 500 ng/ml pertussis toxin (PTX) prevented AA-induced inhibition: in the presence of PTX inhibition was 24.7±6.6% vs 10 nM AVP, as compared to 81.6 ± 4.0% in control groups, i.e in the absence of PTX, N=6. AA had no effect on the cAMP level induced by 5M forskolin. It is concluded that AA inhibits AVP-dependent cAMP accumulation in the rat MTAL by a mechanism which implicates a GTP-dependent protein sensitive to PTX.     

Trypanosoma congolense: the in vitro akinetoplastic induction sensitivity assay

Incubation ofTrypanosoma conglense in diminazene aceturate (Berenil) or isometamidium chloride (Samorin) induced akinetoplastic (AK) forms in vitro. The AK values (expressed in percent) obtained were found to be useful for rapid assessment of relative drug sensitivities. In susceptible clones, AK forms were induced at all drug concentrations tested, whereas in resistant clones they were induced only at higher concentrations. The Berenil-resistant clone exhibited AK values of 0.9%±0.6%–8.9±2% at concentrations of 1–100 g/ml at 4–10 h post-inoculation (p.i.), whereas the Berenil-susceptible clone displayed values of 9.3%±3%–19.2%±5% at 0.1–50 g/ml. Motile trypanosomes were not seen at 100 g/ml at 4 h p.i. or at 10 or 50 g/ml at 10 h p.i. The Samorin-resistant clone showed AK values of 0.5%±0.1%–43%±3% at concentrations of 0.1–100 g/ml at 4 and 10 h p.i., whereas the Samorin-susceptible clone exhibited values of 5.3%±2%–45%±4% at 0.0005–100 g/ml. These results were supported by the findings obtained using a mouse infectivity test.     

Effect of lysolecithin on blood vessel reactivity and of some ethers and esters of glycerophosphocholine and phosphocholine,respectively, on aggregation of rat platelets

Infusion of lysolecithin (LPC; e.g. 88 g/ml for 0.5–1.0 min) did not significantly impair the vasopressor action of norepinephrine (NE), prostaglandin F₂ (PGF₂) and extract of posterior pituitary (EPP) in the isolated perfused hind legs of rats. In other words, vascular smooth muscle behaves differently from the smooth muscle of the guinea-pig small intestine, since, in the latter, contractions evoked by acetylcholine, prostaglandins etc., are inhibited by LPC. Triton X 100 which, by comparison, was used as a detergent effective on the guinea-pig small intestine, depressed the vasopressor effect of NE, PGF₂ and EPP.LPC, at low concentrations (40 mol/l), potentiated (15% max.) ADP-induced platelet aggregation (PA) in rat PRP but, at high concentrations, inhibited PA (IC₅₀=390 mol/l). 2-Hexadecylglycerophosphocholine and its short-chain 1-alkyl ethers, which are structurally related to platelet-activating factor, as well as some long-chain alkanol phosphocholine esters, were somewhat more active than LPC. Dipalmitoyllecithin (4–700 mol/l) was without any effect.     

Effects of inhibitors and ion substitutions on oscillations of cell membrane potential in cells expressing the RAS oncogene

Previous studies revealed that in NIH fibroblasts expressing the ras oncogene but not in other NIH fibroblasts, bradykinin leads to sustained, calcium dependent oscillations of cell membrane potential by repetitive activation of calcium-sensitive K⁺ channels. The present study has been performed to test for ion and inhibitor sensitivity of these oscillations. Both, Lys-bradykinin (kallidin) and bradykinin, but not any shorter peptide tested, maintained the oscillations. The oscillations are abolished in the presence of the K⁺ channel blocker barium (10 nmol/l). The amplitude but not the frequency of the oscillations is dependent on the extracellular potassium concentration. The oscillations are not dependent on the presence of extracellular sodium, bicarbonate or chloride. The oscillations are abolished in the absence of extracellular calcium and their frequency is significantly decreased at reduced extracellular calcium (to 0.2 mmol/l). The oscillations are not inhibited by acute administration of ouabain (0.1 mmol/l), by dimethylamiloride (100 mol/l), furosemide (1 mmol/l) and hydrochlorothiazide (100 mol/l), by cobalt (100 mol/l), zinc (100 mol/l), gadolinium (100 mol/l), verapamil (10 mol/l) and diltiazem (10 mol/l), but are abolished in the presence of 100 mol/l lanthanum, 1 mmol/l cadmium, 10 mol/l nifedipine, 25 mol/l SK & F 96365 and 200 mol/l TMB-8. Stimulation of calcium entry by 10 mol/l ionomycin is frequently followed by oscillations of cell membrane potential even in the absence of bradykinin. In conclusion, in cells expressing the ras oncogene bradykinin leads to sustained activation of calcium channels at the cell membrane, which cause oscillations of the cell membrane potential by triggering intracellular calcium release.     

Criteria for testing the susceptibility ofStreptococcus pneumoniae to cefotaxime and its desacetyl metabolite using 1 μg or 30 μg cefotaxime disks

In vitro susceptibility tests were performed with 350 selected strains ofStreptococcus pneumoniae to evaluate disk diffusion tests with 30 g and 1 g cefotaxime disks. Zones were compared to MICs of cefotaxime with and without its desacetyl metabolite. Cefotaxime was two to eight times more active than desacetyl cefotaxime, but the two compounds were additive when combined in vitro. For 30 g disks, zone size breakpoints were 27 mm, 28–30 mm and 31 mm for resistant, intermediate and susceptible, respectively. For 1 g disks, those zone size criteria were reduced to 13 mm, 14–16 mm and 17 mm. The 30 g disk that is currently available for testing other species can be used for testing pneumococci; however, the 1 g disk has some important advantages.     

Pyramidal tract of the cat: axon size and morphology

Summary The purpose of this work was to determine the number and morphology of pyramidal tract (PT) axons in the cat, using electron microscopy, modern methods of fixation, and computer-assisted morphometric analysis. Sections taken at the level of the medullary pyramids in three animals were fixed and magnified up to 10,000 x to produce photomicrographs. Morphological data were entered into computer files for analysis by tracing axon perimeters on micrographs mounted on a digitizer tablet. The number of axons per PT averaged 415,000, of which 88% were myelinated and 12% were unmyelinated. 90% of the myelinated axons fell in the diameter range 0.5–4.5 m. Axons larger than 9 m diameter accounted for 1% of the total; the largest were 20–23 m. Myelinated axon mean diameter was 1.98 m; because of the skewed distribution, with many small axons and a few very large axons, median diameter was 1.60 m. Size distribution was relatively uniform throughout the PT cross section, with all sizes represented in all regions. However, the more medial regions had a higher proportion of small fibers than the more lateral regions: mean medial diameter was 1.85 m while mean lateral diameter was 2.09 m. Myelin sheath thickness averaged 7.9% of fiber diameter for axons up to 11 m, but was constant at 0.9 m for larger fibers. Myelinated fibers were distorted from the circular shape in cross section, with a mean circularity index (or form factor) of 0.85, which implies that the fibers could swell about 15% without rupture of the cell membrane. Unmyelinated fibers averaged 0.18 m diameter (range 0.05–0.6 m); the largest unmyelinated axons were larger than the smallest myelinated axons. It is concluded that previous work greatly underestimated the number of axons in the cat pyramidal tract.     

Capsaicin preferentially affects small-diameter acutely isolated rat dorsal root ganglion cell bodies

The effects of capsaicin were tested on 221 acutely isolated dorsal root ganglion neurons of the rat, which ranged in diameter from 15 to 55 m. In a subpopulation of these cells, ranging in diameter from 17.5 to 33 m (n=117), capsaicin (1 M) produced an inward shift in holding current that was associated with an increase in membrane conductance in most cells (114 of 117). These effects of capsaicin were reversible upon washout of the drug. Other cells ranging in diameter from 15 to 52.5 m (n=104) were unaffected in this manner by the 1 m concentration of capsaicin. Capsaicin-sensitive cells had, on average, significantly longer duration action potentials and expressed significantly less I_H than capsaicin-insensitive cells. The relatively long duration action potentials and/or small cell body diameter and paucity of I_H observed in most of the capsaicin-sensitive cells is consistent with their representing C- or A-type sensory neurons.     

The control of cell proliferation by preformed purines: A genetic study II. Pleiotropic manifestations and mechanism of a control exerted by adenylic purines on PRPP synthesis

When plated in medium containing 0.5 g/ml coformycin and adenosine (or adenine) fibroblasts were killed, even if pyrimidines were supplied. Measurements of N-formylglycine amide ribonucleotide synthesis showed that lethality is a manifestation of purine starvation. In the case of adenosine kinase deficient cells, growth was restored by hypoxanthine. The adenylic derivatives block only purine biosynthesis, presumably by inhibition of PRPP-amidotransferase. In this same medium, wildtype cells exhibited symptoms of PRPP deprivation: purine and pyrimidine syntheses were both shut off and HGPRT was simultaneously inactivated. The pleiotropic control by adenosine was abolished in adenosineresistant mutants that behaved as PRPP overproducers. These mutations conferred partial resistance to various toxic purine and pyrimidine analogs and preserved HGPRT activity in adenosinecontaining medium. This permits selection against these mutants. Evidence suggesting that adenosine kinase products may fulfill a specific function in the regulation of PRPP synthesis is discussed.Abbreviations AK adenosine kinase - APRT adenine phosphoribosyltransferase - HGPRT hypoxanthine guanine phosphoribosyltransferase - AD adenosine deaminase - OPRT orotate phosphoribosyltransferase - PRA phosphoribosylpyrophosphate amidotransferase - A adenine - rA adenosine - Hx hypoxanthine - dA 2-deoxyadenosine - rU uridine - dT thymidine - PRPP phosphoribosylpyrophosphate - Pala N(phosphonacetyl)-l-aspartate - FGAR -N-formyl-glycinamide ribonucleotide - Amp aminopterin - Amp + dT medium standard ERH medium supplemented with Amp and dT - HC-medium ERH supplemented with 0.5g coformycin/ml.     

On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation

The Ca²⁺ channel subunits _1C-a and _1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba²⁺ current (I _Ba) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 M cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 M PKA and 1 M okadaic acid. The activity of the _1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 M H 89 and was not increased by superfusion with 5 M forskolin plus 20 M isobutylmethylxanthine (IBMX). The _1C-a·₂·₂/ complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 M H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the _1C-a·₂·₂/ channel with 10 M PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca²⁺ current (I _Ca) of cardiac myocytes increased threefold during internal dialysis with 5 M PKA or 25 M microcystin and during external superfusion with 0.1 M isoproterenol or 5 M forskolin plus 50 M IBMX. These results indicate that the L-type Ca²⁺ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.     

Phenotypic differences between induced and spontaneous 2μ-plasmid-free segregants of Saccharomyces cerevisiae

Summary The maximum specific growth rates (_max) of 2 -plasmid-free ([cir°]) segregants of three haploid and one diploid strain of Saccharomyces cerevisiae have been determined and compared with the _max of their 2 -plasmid-containing ([cir ⁺]) progenitors. Two classes of [cir°] strains have been examined: those induced by transformation with a 2 -based recombinant plasmid according to the method of Dobson et al. (1980) and those isolated as spontaneous [cir°] segregants from glucose-limited continuous cultures. The _max of the spontaneous [cir°] segregants was not found to differ significantly from that of their [cir ⁺] parents. In all cases, however, the induced [cir°] strains had a _max which was significantly less than that of their [cir ⁺] counterparts. This effect was particularly marked in the case of the diploid strain where a 34% reduction in _max was observed. The implications of these results are discussed in terms of the effect of the transformation process on host yeast cells.