How important is stimulation of alpha-adrenoceptors for melatonin production in rat pineal glands?

The objective of this study was to determine the role of alpha-adrenoceptors in melatonin production by rat pineal gland. Pineal glands were isolated from adult male rats and maintained in organ baths. The perfusate was sampled every 5 min, stored, and later assayed for melatonin. Exposure to norepinephrine (10 microM) or the beta-adrenoceptor agonist orciprenaline (2-10 microM) increased the glands' production of melatonin. The time courses of melatonin production in response to these agonists were unaffected by the rats' pretreatment in vivo with the alpha-adrenoceptor antagonist prazosin (2 mg/kg i.p., three times). Rats that had had their superior cervical ganglia removed were primed with either orciprenaline (2 mg/kg i.p) or both orciprenaline and phenylephrine (1 mg/kg i.p) 1 hr before decapitation. Exposure of the pineal glands from these rats to orciprenaline evoked melatonin release that was similar in each group. These results lend weight to the suggestion that the marked potentiation by alpha-adrenoceptor agonists of the stimulation of cAMP and N-acetyltransferase (NAT) by beta-adrenoceptor agonists, demonstrated most readily in cultured glands or dispersed rat pinealocytes, does not carry over into significant augmentation of melatonin production in intact pineal glands.

The association between melatonin production and electrophysiology of the guinea pig pineal gland.

Melatonin production by isolated pineal glands from guinea pigs was examined under conditions that affect membrane potential or the firing of action potential-like spikes. In glands from superior cervical ganglionectomized animals, depolarization resulting from increasing extracellular potassium concentration to 100 mM did not initiate melatonin production, and it delayed the response to the beta-adrenoceptor agonist orciprenaline. In glands from intact animals melatonin production was initiated by exposure to 100 mM potassium with a time-course similar to the response to orciprenaline. A proportion of this response was propanol resistant, suggesting that the normal control of melatonin production may involve a neurotransmitter in addition to norepinephrine. Exposure to verapamil or nifedipine, or removal of extracellular calcium, previously shown to eliminate action potential-like spikes, did not substantially affect the increase in melatonin production induced by orciprenaline. Phenylephrine, which stimulates spiking, produced only a slight increase in melatonin production. It is concluded that the depolarization and the spiking are not closely related to the stimulation of melatonin production, but may relate principally to the secretion of a substance other than melatonin.

Assessment of statistical procedures used in papers in the Australian Veterinary Journal.

One hundred and thirty-three papers (80 Original Articles and 53 Short Contributions) of 279 papers in 23 consecutive issues of the Australian Veterinary Journal were examined for their statistical content. Only 38 (29%) would have been acceptable to a statistical referee without revision, revision would have been indicated in 88 (66%), and the remaining 7 (5%) had major flaws. Weaknesses in design were found in 40 (30%), chiefly in respect to randomisation and to the size of the experiment. Deficiencies in analysis in 60 (45%) were in methods, application and calculation, and in the failure to use appropriate methods for multiple comparisons and repeated measures. Problems were detected in presentation in 44 (33%) of papers, with insufficient information about the data or its statistical analysis and presentation of statistics (appropriate missing or inappropriate shown) the main problems. Conclusions were considered to be inconsistent with the analysis in 35 (26%) of papers, due mainly to their interpretation of the results of significance testing. It is suggested that statistical refereeing, the publication of statistical guidelines for authors and statistical advice to Animal Experimentation Ethics Committees could all play a part in achieving improvement.

Melatonin production by the guinea pig pineal gland in vitro.

The fluid bathing pineal glands isolated from guinea pigs was collected serially and its melatonin content was estimated. Production was found to be high (500 pg/5 min) soon after isolation of the gland: it declined exponentially to a maintained lower level (50 pg/5 min) with a half-time of 16-20 min. A rapid increase could be produced by exposure of the gland to the beta-adrenoceptor agonist orciprenaline or by stimulating it electrically. The time course and the extent of the responses to either form of stimulation were similar in glands that had been taken in the morning, at dusk or in mid-dark: the prestimulation peak was lower from glands taken at dusk. Thus the pineal gland of the guinea pig is capable of responding rapidly to stimulation of its beta-adrenoceptors at any time. These responses parallel the depolarization observed intracellularly in this species when the pineal gland is stimulated electrically or exposed to beta-adrenoceptor agonists.

Effect of isoprenaline on the output of melatonin from fetal and newborn lamb pineal glands in vitro.

We have measured the output of melatonin in vitro from the pineal glands of lambs in late gestation (135-143 days) and the early newborn period (4-8 days). The basal output of melatonin/mg pineal tissue was significantly lower in the fetal group (16.3 +/- 3.9 pg melatonin.(30 min)-1.mg-1) than in the newborn group (148.1 +/- 43.6 pg melatonin.(30 min)-1.mg-1). Addition of the beta-adrenergic agonist, isoprenaline (10(-5) mol/l) significantly stimulated melatonin output from both the fetal (to 24.9 +/- 3.9 pg melatonin.(30 min)-1.mg-1) and newborn pineals (to 247.7 +/- 67.3 pg melatonin.(30 min)-1.mg-1). Pineals from newborn lambs produced 9-10-fold more melatonin than the fetal pineal glands, before, during and after addition of isoprenaline. These results show that functional beta-adrenergic receptors are present before birth on the sheep pinealocyte and that there is a marked increase in the melatonin synthetic capacity of the pineal in the newborn period.

Passively propagated spikes from the soma of cells in pineal gland of guinea pigs.

The membrane properties of cells within the pineal gland of guinea pigs were studied using intracellular electrophysiological techniques. The electrotonic responses to intracellular current injection decayed with a single exponential in approximately 60% of cells but was preceded by a quicker component in the remainder. The membrane time constant was 2.8 ms. Depolarization beyond -29 mV activated an outward current that reversed at around the value of the resting potential. Hyperpolarization activated a slow inward current. Spikes occurred in response to activation of alpha-adrenoceptors. They were resistant to tetrodotoxin but were abolished by nifedipine and verapamil, suggesting that calcium carries the current during their upstroke. Spikes could not be evoked by depolarizing current pulses of 1-ms to 2-s duration. The responses to hyperpolarizing current steps or voltage-clamp steps applied during the peak of spikes evoked by nerve stimulation were indistinguishable from the responses to those applied between spikes. During nerve stimulation, fluctuations were observed in the current trace of cells under voltage clamp, indicating that the spikes could not be voltage clamped successfully. It is concluded that the spikes occurring in response to nerve stimulation are generated on the processes of the pinealocytes and are passive in the soma.

Two types of cells with central innervation in pineal gland of guinea pigs.

Cells within pineal glands isolated from young, male guinea pigs were impaled with intracellular microelectrodes and their responses to stimulate the nerve supply to the gland were studied. Two types of cells were identified. The response of cells of type I was a depolarization on which spikes were superimposed. Blockers of alpha-adrenoceptors abolished the spikes while beta-adrenoceptor blockers reduced the depolarization to 27%, leaving a small tetrodotoxin-sensitive depolarization. After bilateral removal of the superior cervical ganglia (SCG) the beta-mediated depolarization was not observed while the spikes and the smaller depolarization persisted. The response of cells of type II was an initial large, transient depolarization followed by a smaller depolarization. Both components were reversibly blocked by tetrodotoxin. The only agents found to have any effect on these cells were oxytocin, vasopressin, and vasotocin. These peptides caused depolarization similar in amplitude to the larger response to nerve stimulation, although more prolonged. The large depolarization was not observed following ganglionectomy, but the smaller one persisted. It is concluded that cells of type I and II both receive inputs from nerves whose cell bodies lie in the SCG. Cells of both types are also innervated through another pathway.

Effect of duration of stimulation and pH on energetics of rabbit rectococcygeus muscle.

The mechanical and energetic performance of the rabbit rectococcygeus preparation have been studied at 27 degrees C. Energy flux has been measured either myothermically (heat production) or by recording the O2 consumption and lactate production of the muscle. The maximal active-to-basal O2 consumption ratio was 2.42. The peak oxidative energy flux was 3.5 mW/g, and the peak initial rate was about 5.0 mW/g. We could find no evidence for an increment in lactate production associated with mechanical activity. The effects of increasing the stimulus duration on the "economy" of force maintenance was assessed by plotting the heat-to-tension ratio against duration. For stimulus periods up to 20 s there was no evidence of a change in the economy: the slope of the relationship was similar in myothermic and O2 consumption experiments with split muscles. When total heat production was plotted against the stress-time integral for stimulus periods up to 60 s at both pH 7.4 and 6.7, there was no change in energy usage in the longer duration tetani (2-60 s). However, the reduction in pH, which had minimal mechanical effects other than prolonging relaxation, did slow the rate of evolution of recovery heat (time constant doubled) and reduced the rate of O2 consumption. It is concluded that the rectococcygeus preparation cannot make use of the energy-saving mechanisms that operate in arterial and several invertebrate smooth muscles.

The three-ion test for the identity of the ionophores mediating the hyperpolarizing response to cholinomimetics in neurones of Helix aspersa.

1. The hyperpolarizing response investigated is mediated by Cl- ions. Additional anions NO-3 and BrO-3 were introduced into the perfusate to produce a system with 3 permeant anions. 2. Under such circumstances the reversal potential (Er) becomes a means of discriminating between the molecular mechanisms invoked by different agonists that produce an increase in anionic conductance. 3. A statistically significant difference of 0.49 mV was detected between the Er for acetylcholine and those for acetyl-beta-methyl choline and tetramethylammonium (acetylcholine more negative). 4. This difference was not significantly affected by changing to a different 3-ion solution. 5. It is argued from theoretical grounds that a change in this difference is to be expected when molecular mechanisms differ; alternative explanations for the difference are suggested.

The effects of ACTH on adrenal steroidogenesis and blood corticosteroid levels in the echidna (Tachyglossus aculeatus).

1. The effects of short-term (S.T., 30 min) and long-term (L.T., 4 days) administration of ACTH on peripheral blood corticosteroid levels and on in vitro steroidogenesis were investigated. 2. Control levels of cortisol, corticosterone and aldosterone were 58 +/- 12, 130 +/- 26 and 10 +/- 6 (SEM) ng/100 ml respectively. 3. Corticosterone was 70% higher after S.T. and 150% higher after L.T., when cortisol was 800% higher. 4. Adrenal homogenates from control echidnas converted [14C]progesterone predominantly to 11-deoxycorticosterone (45%) and 11-deoxycortisol (12%). 5. After L.T. the principal product was corticosterone (25%), but S.T. had no effect. 6. In control echidnas the Km and V for 11 beta-hydroxylation of 11-deoxycorticosterone were 20 microM and 2.8 rho mol/min/mg respectively. After L.T. V increased to 10 rho mol/min/mg.