David Sugden

Activators of Protein Kinase C Act at a Postreceptor Site to Amplify Cyclic AMP Production in Rat Pinealocytes

Abstract: Activation of α1‐adrenoceptors appears to amplify β‐adrenergic stimulation of cyclic AMP (CAMP) accumulation in rat pinealocytes severalfold by a mechanism involving activation of a Ca2+‐, phospholipid‐dependent protein kinase (protein kinase C). The mechanism of action of protein kinase C was investigated in this report using intact cells. Activation of protein kinase C with 4β‐phorbol 12‐myristate 13‐acetate (PMA; 10−7M) or the α1‐adrenergic agonist phenylephrine (PE; 10−6M) did not inhibit cAMP efflux in β‐adrenergically stimulated cells. The amplification of the β‐adrenergic cAMP response by these agents also occurred in the presence of isobutylmethylxanthine (10−3M) and Ro 20–1724 (104‐M), an observation suggesting that inhibition of cAMP phosphodiesterase activity is not the mechanism of action. Furthermore, although PMA (107‐ M) caused a sixfold increase in the magnitude of the cAMP response to isoproterenol, it did not alter the EC50 of the response (1.7 × 10−8M), a result indicating that protein kinase C activation does not alter β‐adrenoceptor sensitivity. The cAMP response following cholera toxin pretreatment (60–120 min) was rapidly and markedly enhanced by a,‐adrenergic agonists (cirazoline > PE > methoxamine), by phorbol esters (PMA > 4β‐phorbol 12,13,‐dibutyrate >> 4α‐phorbol 12,13‐didecanoate), and by synthetic diacylglycerols (1,2‐dioctanoylglycerol > 1‐oleoyl 2‐acetylglycerol >> diolein). The cAMP response to forskolin (10−5M) was also increased by PE (3 × 10−6M) and PMA (10−7M). Together, these observations indicate that protein kinase C activation amplifies β‐adrenergic stimulation of pinealocyte cAMP production at a site beyond the receptor, perhaps on a regulatory guanine nucleotide binding protein or adenylyl cyclase itself.

Regulation of Rat Pineal Hydroxyindole-O-Methyltransferase in Neonatal and Adult Rats

The relative importance of neural, and some nonneural, mechanisms in the control of pineal hydroxyindole‐O‐methyltransferase (HIOMT) activity during development and in the adult rat was studied. In neonatal rats, guanethidine‐treatment, bilateral superior cervical ganglionectomy (SCGX), or exposure to constant light did not prevent the initial appearance of HIOMT activity, indicating that neural stimulation of the gland is not essential for the development of HIOMT activity. In adult rats, decentralization or removal of the SCG led to a slow fall in HIOMT activity, to about 30% of control activity, indicating that the enzyme is largely under neural control. Additionally, adrenalectomy or hypophysectomy had no effect on HIOMT activity, refuting the suggestion that adrenal and/or gonadal steroids are of major importance in the regulation of this enzyme. The fall in activity of the enzyme after SCGX or exposure to constant light probably does not represent a shift in the Km of the enzyme nor the selective disappearance of a distinct molecular species. Similar changes in HIOMT activity and cyclic GMP responsiveness occur in response to alterations in the length of the daily dark period, adding further evidence to our earlier speculation that there may be a functional relationship between these two.

See‐Saw Signal Processing in Pinealocytes Involves Reciprocal Changes in the α1‐Adrenergic Component of the Cyclic GMP Response and the β‐Adrenergic Component of the Cyclic AMP Response

Abstract: Pineal cyclic AMP and cyclic GMP are regulated by norepinephrine (NE) acting through α1‐‐ and β‐adrenoceptors. β‐Adrenergic stimulation appears to be an absolute requirement and a,‐adrenergic activation amplifies β‐adrenergic stimulation of the cyclic AMP response 10‐fold and the cyclic GMP response 100‐fold, respectively. Chronic deprivation of adrenergic stimulation, due to exposure to constant light (LL) or by surgical denervation, enhances the cyclic AMP response and diminishes the cyclic GMP response as compared to control animals in a 10:14 light/dark (LD) cycle. This phenomenon is termed see‐saw signal processing. In the current study we find these changes do not reflect shifts in the time course or Ka of these responses. Dose‐response studies indicate the p‐adrenergic component of cyclic AMP stimulation is enhanced and the α1‐adrenergic component of cyclic GMP stimulation is diminished in LL pinealocytes. Several observations indicate these changes may reflect alterations in Ca2+ ‐sensitive postreceptor mechanisms.

Alpha-adrenergic potentiation of beta-adrenergic stimulation of rat pineal N-acetyltransferase. Studies using cirazoline and fluorine analogs of norepinephrine.

Recent evidence indicates that melatonin production is controlled by norepinephrine acting via alpha 1-and beta 1-adrenoceptors on pinealocytes; activation of alpha 1-adrenoceptors appears to potentiate the effects of beta 1-adrenoceptor activation. However, alpha-adrenergic potentiation of beta 1-adrenergic activation has been demonstrated with only one alpha-adrenergic agonist. For this reason, this issue was reinvestigated using two other alpha-adrenergic agonists, 6-fluoronorepinephrine and cirazoline. Both compounds, which were found to have a high affinity for pineal alpha 1-adrenoceptors, potentiated the stimulatory effects of isoproterenol on pineal N-acetyltransferase. 6-Fluoronorepinephrine also potentiated the stimulation of N-acetyltransferase activity produced by another beta-adrenergic agonist, 2-fluoronorepinephrine. These findings support the hypothesis that pineal N-acetyltransferase activity is regulated by norepinephrine acting through both alpha 1- and beta 1-adrenoceptors.

Adrenergic stimulation of rat pineal hydroxyindole-O-methyltransferase

Abstract Pineal hydroxyindole-O-methyltransferase (HIOMT) activity is known to decline slowly in rats exposed to constant light. We have found that long-term, in vivo isoproterenol treatment prevents and reverses this decline. Similarly, enzyme activity declines slowly in rats after bilateral superior cervical ganglionectomy. We have also found this effect can be prevented by long-term, in vivo treatment with norepinephrine or isoproterenol. These results, the first demonstration that HIOMT activity can be stimulated by treatment with adrenergic agonists, indicate that the neurotransmitter involved in the adrenergic control of HIOMT activity is norepinephrine.

Age‐Associated Changes in Pineal Adrenergic Receptors and Melatonin Synthesizing Enzymes in the Wistar Rat

The nocturnal stimulation of pineal melatonin synthesis and elevation of serum melatonin is known to be reduced in old age in several species. In Wistar rats the capacity of the β‐adrenoceptor to develop supersensitivity (increase in Bmax) during the light period of the diurnal light/dark cycle is lost during maturation (3–6 months) rather than old age. Further, the present study shows that neither the α1‐ nor β‐adrenoceptor density of the pineal declines as rats age. Pineal hydroxyindole‐O‐methyltransferase activity does fall (17–55%) in rats after 18 months of age, but nocturnal pineal arylalkylamine N‐acetyltransferase activity is not significantly altered. Thus, from examination of these parameters across the life span of the rat, it seems likely that the reported reduction in serum melatonin in old animals is related to a reduced capacity of the pineal to synthesize melatonin, rather than an altered responsiveness of the gland to neural stimulation.

Regulation of Rat Pineal α1‐Adrenoceptors

Some aspects of the physiological regulation of the pineal α1‐adrenoceptor have been studied using the selective, high‐affinity ligand [125I] iodo‐2‐[β‐(4‐hydroxyphenyl)ethylaminomethyl]tetralone ([125I]HEAT). Pineal glands taken from rats housed in a diurnal lighting cycle showed no circadian rhythm in the number of specific [125I]HEAT binding sites, although a characteristic rhythm in pineal melatonin was seen. It was established that the pineal α1‐adrenoceptor is under neural control because interruption of neural stimulation of the pineal by bilateral superior cervical ganglionectomy (SCGX) or by exposing rats to constant light for 3 weeks doubled receptor density but did not change affinity for [125I]HEAT. Administration of various α1‐adrenoceptor agonists either acutely (i.p. injection) or chronically (s.c. infusion) did not alter the number of specific [125I]HEAT binding sites. Together these results indicate that the pineal α1‐adrenoceptor, like the pineal β‐adrenoceptor, is regulated by sympathetic nerve activity, probably through the physiological release of the neurotransmitter norepinephrine. However the absence of a circadian rhythm in α1‐adrenoceptor number and lack of down‐regulation by adrenergic agonists imply different mechanisms of regulation.

Ovine Pineal Indoles: Effects of l-Tryptophan or l-5-Hydroxytryptophan Administration

Abstract: l.‐5‐Hydroxytryptophan (l‐5‐HTP) (20 or 200 mg/kg i.p.) but not l‐tryptophan (500 mg/kg i.p.) loading substantially increases serum melatonin in sheep. In the present study we examined the effects of these compounds on pineal serotonin and six serotonin metabolites. l‐Tryptophan failed to increase 5‐hydroxytryptamine (5‐HT; serotonin) or any of its metabolites despite a fivefold increase in pineal tryptophan. In contrast, l‐5‐HTP loading produced a marked increase in pineal 5‐HT and its metabolites, including N‐acetylserotonin (NAS) and melatonin, indicating that an increased synthesis of melatonin is responsible for the increased serum melatonin concentration after loading with this precursor. No change in pineal indoleamine N‐acetyltransferase (NAT) activity was seen. These results are consistent with the suggestion that, during daytime in the sheep, 5‐HT availability may limit the production of melatonin.

Development of the rat pineal #x003B1;1-adrenoceptor

Abstract Pineal α1-adrenoceptors in rats from 19 days of gestation until 11 months of age were studied using [125I]iodo-2-[β-(4-hydroxyphenyl)ethylaminomethyl]tetralone ([125I]HEAT). The number of specific [125I]HEAT binding sites increased markedly between 18 days of gestation (101.7 ± 13.1fmol/mg protein) and 10 days of age (336.2 ± 34.3fmol/mg protein). A significant decline occurred after 1 month of age. A saturation study showed similar change in receptor density with age (Bmax; 20 days of gestation, 130.5 fmol/mg protein; 35 days old, 288.1 fmol/mg protein) but no difference in Kd(58.4pM at both− 1and+ 35days). The developmental appearance of the pineal α1-adrenoceptor and the decline in its density with age are remarkably similar to changes reported for pineal β-adrenoceptors.

Measurement of Tryptophan Hydroxylase Activity in Rat Pineal Glands and Pinealocytes Using an HPLC Assay With Electrochemical Detection

A method for measuring tryptophan hydroxylase activity by assaying the product 5‐HTP using high‐performance liquid chromatography (HPLC) with electrochemical detection is described. A nocturnal elevation (80%) in rat pineal gland tryptophan hydroxylase activity was detected. Experiments on isolated rat pinealocytes with the protein synthesis inhibitor cycloheximide indicate that tryptophan hydroxylase turns over rapidly in these cells. This method will be valuable in studies of the adrenergic mechanisms regulating pineal tryptophan hydroxylase activity.