Studies on the micro‐injection of various substances into crab muscle fibres

SUMMARY1. In single striated muscle fibres from the barnacle Balanus nubilu8 tension development following axial injection of caffeine (50 mM in 150 mM-KCl, pH 7.1) was used as an index of releasable Ca. It was shown that fibres incubated in 0 Ca (Na replaced) salines for up to 400 min gave ca. 15 % of the control tension response. Inclusion of 1 mM-La in the 0 Ca (Na) saline significantly reversed this decline.2. Estimates of the total fibre calcium assayed under the same experimental conditions indicated a 67 % loss of fibre Ca in 0 Ca, and only a 37 % loss in La-0 Ca media. No correction was made for the loss of calcium from the extracellular space.3. Experiments with 45Ca indicated that the efflux of calcium from this preparation was inhibited by 1 mM-La externally and that this effect was still significant even in the presence of 0 Ca (Na) salines. The caffeinestimulated efflux of Ca was also reduced by ca. 70 % in the presence of 1 mm La saline externally.4. The influx and efflux of 14C caffeine were shown to be rapid, and apparently passive. The diffusion coefficient for caffeine following intracellular injection was 2-4 + 0-2 x 10-6 cm2 sec-' at 18-22°C.5. There was no significant loss of 140La over a period of 2 hr following axial micro-injection into the fibres.6. In 140La uptake experiments there was a progressive increase in the La space over 10-5 hr, in contrast to the results with [3H]inulin, whose uptake saturated in ca. 1-5 hr. The probability of surface binding and the precipitation of La salts in the extensive extracellular space was suggested as an explanation.7. It is concluded that internal Ca within the sarcoplasmic reticulum, and not cleft or extracellular Ca is the most significant source for these caffeine-induced contractions. Fluxes across the surface membrane can however alter the internal Ca stores over longer periods of time.

Section: Discussionmentioning

confidence: 72%

Section: Introductionmentioning

confidence: 99%

Caffeine and the contractility of single muscle fibres from the barnacle Balanus nubilus

Ashley

Ellory

Griffiths

1977

“…However, by inserting a longitudinal Ag-Ag(l wire down a single fibre using the method of Caldwell & Walster (1963) the noise in the recording system can be reduced to less than 0-5 #V r.m.s. over a kilocycle bandwidth, and a continuous random discharge of depolarizing min.…”

Section: L-glutamate Voltage Noisementioning

confidence: 99%

“…Single muscle fibres were dissected and cannulated in a similar way to that described by Caldwell & Walster (1963) with the exception that the fibres were mounted horizontally. The recording electrode, an Ag-AgCl wire (250 sm in diameter), was inserted, using a micromanipulator, through the cannula into the muscle fibre.…”

Section: Glutamate Noise 207mentioning

confidence: 99%

On the elementary conductance event produced by L‐glutamate and quanta of the natural transmitter at the neuromuscular junctions of Maia squinado.

Crawford¹,

McBurney²

1976

1. The membrane potential of giant muscle fibres of Maia squinado was measured with an intracellular wire electrode. On applying L‐glutamate to the fibre the cell deplorized and fluctuations of the membrane potential around its mean level‐‐glutamate noise‐‐were seen. 2. The variance of the glutamate voltage noise is proportional to the mean level of depolarization. The noise can be regarded as being caused by numerous exponentially decaying elementary voltage events about 5 X 10(‐10) V in amplitude. The miniature excitatory junctional potential (min.e.j.p.) is approximately 6000 times the amplitude of the elementary voltage event produced by L‐glutamate. 3. The power spectrum of glutamate voltage noise is a Lorentzian with a half‐power frequency of approximately 20 Hz. 4. Min. e.j.p.s. decay exponentially with a time constant that coincides with the average lifetime of the elementary glutamate voltage event. 5. When glutamate is applied locally to a spot where extracellular min. e.j.p.s. can be recorded with a focal glass pipette, extracellular glutamate noise is seen. Glutamate noise could not be detected from elsewhere on the fibre. 6. The variance of the extracellular noise is proportional to the mean extracellular potential, and its power spectrum is a Lorentzian with a half‐power frequency of about 110 Hz. 7. The extracellular min. e.j.p.s decay exponentially with a time constant that coincides with average lifetime of the elementary glutamate current event. 8. It is suggested that the decay of the quantal currents flowing at the excitatory junction is limited by the closure of the conductance channels in the post‐synaptic membrane and not by the relaxation of the transmitter concentration in the synaptic cleft.

“…The temperature of the ASW was maintained at 12 'C. Single muscle fibres were isolated by dissection from the three pairs of depressor muscle bundles and cannulated in the same way as crab muscle fibres (Caldwell & Walster, 1963). A 50-80 mg weight was attached to the tendon end of the cannulated fibre.…”

Section: Methodsmentioning

confidence: 99%

Stimulation by injected guanosine triphosphate of the sodium efflux in barnacle muscle fibres.

Bittar¹,

Nwoga²

1982

1. A study has been made of the mechanism by which injected disodium GTP stimulates the ouabain‐insensitive Na efflux in single muscle fibres from the barnacle, Balanus nubilus. 2. Injection of GTPNa2 causes a stimulatory response which is usually transitory and almost completely reversed by injecting MgCl2 (but not KCl). 3. Injected 5'‐guanylylimidodiphosphate, Gpp(NH)p, mimics this action of GTP but the reversal seen with injected Mg2+ is less pronounced. 4. (i) Pre‐treatment of these fibres with verapamil reduces the size of the stimulatory response to GTP and Gpp(NH)p. (ii) Pre‐injection of protein kinase inhibitor (PKI) or regulatory subunits reduces the response as well. (iii) Pre‐treatment with imipramine or trifluoperazine reduces the response to injected GTP; in combination with verapamil, a greater reduction in response is seen. 5. Injection of EDTA leads to a stimulatory response which is transitory. This response is largely abolished by verapamil. 6. Injection of cholera toxin leads to a sustained stimulatory rather than a transitory response. GTP or Gpp(NH)p when injected following peak stimulation by cholera toxin leads to a moderate sustained stimulation. 7. These results support the view that the stimulatory response to injected GTPNa2 is the result of activation of Ca2+ channels and of increased availability of GTPMg and that these two conditions bring about activation of adenylate cyclase and hence activation of cyclic AMP‐protein kinase by newly formed cyclic AMP.