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PubMed1989

Antinociceptive effect of agonists microinjected into the anterior pretectal nucleus of the rat.

Electrical stimulation at many sites within the pretectal complex and adjacent structures of the rat dorsomedial thalamus yields antinociception. It is documented that no other site in this region evokes antinociception longer lasting than that obtained by stimulation of the anterior pretectal nucleus (APtN). The effects of agonists injected into different nuclei of the dorsomedial thalamus on the tail-flick reflex of rats in response to noxious heat was examined. All animals were submitted to intracerebral electrical stimulation and microinjection of agonists. It was confirmed that strong and long lasting antinociception followed brief (15 s), low intensity (35 microA rms) stimulation of the APtN. In addition, L-glutamate (3.5 and 7.0 micrograms), morphine (1.0 and 5.0 micrograms), and 5-hydroxytryptamine (5-HT; 2.5 and 5.0 micrograms), but not acetylcholine (5.0 micrograms), carbachol (2.5 micrograms), norepinephrine (5.0 micrograms), or dopamine (5.0 micrograms), induced dose-dependent antinociception when microinjected into the APtN. The effect of 5-HT was fully depressed by pretreating animals with methysergide (5 mg/kg, i.p.). A survey of the sites from which morphine and 5-HT induce antinociception revealed that in no site of the dorsomedial thalamus did the effect last longer than after microinjection into the APtN. It is concluded that antinociception evoked by stimulation of the APtN depends on the activation of neuronal cell bodies in the nucleus, and that 5-HT and endogenous opioids may play a physiological role as neurotransmitters mediating antinociception in the APtN.

Analgesia↗

PubMed1989

Glycine, like glutamate, microinjected into the nucleus tractus solitarii of rat decreases arterial pressure and heart rate.

Glycinergic mechanisms have been implicated in central cardiovascular regulation. However, the inhibitory amino acid's role in the nucleus tractus solitarii (NTS), the site of termination of cardiovascular afferents, has not been clarified. Thus, we sought to determine if the microinjection of glycine into the NTS alters arterial pressure and heart rate. Microinjections of glycine, like glutamate, confined to the NTS decreased arterial pressure and heart rate in a neurally mediated, dose-dependent manner. The glycine antagonist strychnine completely blocked these effects of glycine but did not itself alter arterial pressure or heart rate, or interfere with the baroreceptor reflex. The acute hypotensive, bradycardic response to glycine was followed by a period during which glycine essentially eliminated the cardiovascular responses to the microinjection into the NTS of glutamate, an amino acid reputed to be a transmitter in the baroreceptor reflex arc. These data suggest that glycine is involved in cardiovascular regulation by the NTS but do not support its being an integral transmitter in the baroreceptor reflex.

Animals↗

PubMed1989

A critical role of the posterior hypothalamus in the mechanisms of wakefulness determined by microinjection of muscimol in freely moving cats.

In order to determine critical sites within the hypothalamus responsible for the induction and maintenance of wakefulness (W), we performed microinjections of muscimol, a potent gamma-aminobutyric acid (GABA) agonist, in various lateral hypothalamic regions of freely moving cats. We found that bilateral injections of a small amount of muscimol (0.1-1.0 micrograms/0.5 microliters) in the preoptic and anterior hypothalamus and rostral mesencephalic tegmentum resulted in increased vigilance and insomnia. In contrast, microinjections of muscimol in the middle and anterior parts of the posterior hypothalamus induced long-lasting behavioral and electroencephalographic signs of sleep with short latency. The hypersomnia was characterized by a significant increase in both light and deep slow wave sleep (SWS), and a nearly complete suppression of paradoxical sleep (PS). Animals with muscimol microinjections in the ventrolateral part of the posterior hypothalamus, however, exhibited increased SWS followed by a significant increase in PS. When injected into the posterior hypothalamus of insomniac cats pretreated with p-chlorophenylalanine (PCPA), muscimol induced not only SWS but also PS with short latency. The present data thus support the hypotheses that the posterior hypothalamus plays a critical role in the mechanisms of W and that sleep might result from functional blockade of the hypothalamic waking center.

Animals↗

PubMed1990

Microinjection of TRH analogs into the raphe pallidus stimulates gastric acid secretion in the rat.

The effects of microinjection of the stable thyrotropin-releasing hormone (TRH) analog, RX 77368, [pGlu-His-(3,3'-dimethyl)-Pro-NH2] into the raphe pallidus on gastric acid secretion were studied in urethane-anesthetized rats with gastric fistula. RX 77368 microinjected into the raphe pallidus at doses of 0.07, 0.7 and 7.7 pmol induced a dose-dependent net stimulation of gastric acid secretion (7 +/- 4, 50 +/- 7 and 61 +/- 12 mumol/h respectively). The peak acid response was reached within 30 min and returned to basal level 90 min post-injection. The stimulatory effect was abolished by bilateral cervical vagotomy and pirenzepine pretreatment (1 mg/kg, i.v.). RX 77368 (7.7 pmol) microinjected into the inferior olive or pyramidal tract induced smaller or no gastric acid secretory response. These results demonstrate that chemical stimulation of the raphe pallidus increases gastric acid secretion through vagal pathways and peripheral muscarinic receptors. These data suggest that the nucleus raphe pallidus may be involved in vagal modulation of gastric acid secretion in the rat.

Animals↗

PubMed1990

Inhibition by carbachol microinjections of presumptive cholinergic PGO-on neurons in freely moving cats.

The effects of microinjections of a cholinergic agonist, carbachol (0.2 micrograms/0.2 microliters), were examined on a population of presumptive cholinergic mesopontine PGO-on neurons that presents a tonic pattern of discharge during waking and exhibits short spike bursts preceding the onset of dorsal lateral geniculate PGO waves during paradoxical sleep and slow wave sleep just prior to it. PGO-on neurons were activated antidromically by the stimulation of the dorsal lateral geniculate, pulvinar and/or medial and intralaminar thalamic nuclei. They were all characterized by a long spike duration and a slow conduction velocity. Microinjections of carbachol near unit recording sites in freely moving cats induced a complete suppression of the spontaneous tonic activity during waking, but did not suppress the spontaneous phasic burst activity during sleep. Carbachol microinjections also resulted in a marked reduction in responsiveness of PGO-on neurons to orthodromic stimulation. These spike depressant effects lasted for approximately 90-120 min and were reversed completely by a local or systemic administration of atropine sulfate. These findings point to a direct inhibition of central cholinergic PGO-on neurons via a muscarinic autoreceptor and a difference in the mechanisms underlying the generation of tonic and phasic burst activity of PGO-on neurons occurring during waking and sleep.

Animals↗

PubMed1990

Flight and immobility evoked by excitatory amino acid microinjection within distinct parts of the subtentorial midbrain periaqueductal gray of the cat.

Unilateral microinjections of the excitatory amino acid, D,L-homocysteic acid (DLH) made in the lateral and ventrolateral parts of the subtentorial (A 1.0-P 1.5) midbrain periaqueductal gray (PAG) of the freely moving cat evoked two distinct patterns of coordinated somatic changes. When DLH injection (80 nmol) was made within the lateral part of the subtentorial PAG it evoked a flight reaction, characterized by strong locomotion (running) and multiple jumps. This flight reaction was quite distinct from the defensive threat display previously described following DLH microinjection in the lateral part of the pretentorial PAG. When DLH injection (80 nmol) was made in the subtentorial PAG region, ventrolateral to the aqueduct, it elicited a cessation of both spontaneous locomotion and general movements (e.g. licking, scratching, grooming, head and limb movements), a reaction termed immobility. The subtentorial PAG regions from which flight and immobility were evoked are seemingly identical to the lateral and ventrolateral subtentorial PAG regions in which hypertensive and hypotensive reactions have been evoked previously by DLH microinjection. The present results together with our previous studies suggest that: (1) the lateral PAG of the cat contains at least two, topographically separable neuronal pools, which mediate different types of defense reactions (i.e. threat display--lateral part of the pretentorial PAG; flight reaction--lateral part of the subtentorial PAG); and (2) excitation of neurons in the ventrolateral PAG alters autonomic and somatic functions, but in a direction opposite to that of lateral PAG neurons, namely decreased somatomotor activity and hypotension.

Animals↗

PubMed1990

The antinociceptive effects of SCH-32615, a neutral endopeptidase (enkephalinase) inhibitor, microinjected into the periaqueductal, ventral medulla and amygdala.

The local effects of SCH-32615, an inhibitor of enkephalinase (EC 3.4.24.11) on the hot-plate (HP) and tail-flick (TF) responses were examined following unilateral intracerebral microinjection into the periaqueductal brain (PAG), the medial ventral medulla (VM) and bilateral microinjection into the amygdala (AM) of the rat. In the PAG and VM, SCH-32615 resulted in a dose-dependent increase in HP and TF response latencies over a dose range of 1-30 micrograms with the ED50 values (micrograms) being PAG-TF = 17; PAG-HP = 11; VM-TF = 7; VM-HP = 6. In the AM, dose-dependent increases were only observed on the HP. (ED50 (micrograms) HP = 17). Peak effects were observed within 10 min and response latencies remained elevated for 45-60 min. Injections of SCH-32615 at sites outside of the PAG or VM were considerably less effective. All antinociceptive effects were antagonized by naloxone (1 mg/kg, i.p.). Twenty-four hours following the microinjection of beta-funaltrexamine (an irreversible opioid antagonist) into the PAG or the VM, the effects of SCH-32615 in the PAG were virtually abolished while in the VM, its effects were only moderately reduced. These data suggest that in the presence of a strong thermal stimulus, the behavioral response is subject to a tonically active or stimulus-evoked modulation by the local release in the PAG, VM and AM of an agent, presumably an enkephalin peptide, the degradation of which is altered by enkephalinase inhibition.

Amygdala↗

PubMed1991

Microinjection of the dopamine antagonist cis-flupenthixol into the MPOA impairs copulation, penile reflexes and sexual motivation in male rats.

Microinjection of the dopamine antagonist cis-flupenthixol into the medial preoptic area was previously shown to impair male rat copulatory behavior. The present experiments provide further evidence of cis-flupenthixol's inhibitory effects on male sexual behavior. Following microinjections of moderate to high doses of cis-flupenthixol, males exhibited slower copulatory rates and fewer ejaculations in copula, fewer ex copula erections and penile movements, and reduced sexual motivation in an X-maze. Locomotion in the X-maze was not significantly affected. Microinjections of the inactive isomer trans-flupenthixol produced no change in any behavioral measure, indicating that cis-flupenthixol's effects were receptor mediated. We suggest that dopamine receptors in the MPOA influence copulation primarily by regulating reflexive and motivational factors, but not locomotion.

Animals↗

PubMed1993

Pressor response to microinjection of clonidine into the hypothalamic paraventricular nucleus in conscious rats.

We have reported that intracerebroventricular (i.c.v.) injection of clonidine causes pressor response in conscious rats. To determine the effective brain site, cardiovascular responses induced by unilateral microinjection of clonidine into various hypothalamic nuclei of conscious rats were studied. Microinjection of clonidine (5-20 micrograms/0.5 microliter) into the paraventricular nucleus (PVN) of conscious rats dose-dependently produced a long-lasting pressor response with a decrease in heart rate, which mimicked the response to i.c.v. injection of clonidine. However, clonidine (10 micrograms) injection into various hypothalamic nuclei (anterior, posterior, ventromedial and dorsomedial nucleus) caused a small or no pressor response. In anesthetized rats, clonidine injected into the PVN induced a long-lasting depressor response concomitant with bradycardia. PVN pretreatment with the alpha 2-adrenoceptor antagonist, yohimbine (1 and 10 micrograms), dose-dependently inhibited the pressor response to PVN injected clonidine, but the alpha 1-adrenoceptor antagonist, prazosin (1 microgram), had no significant effect. Central (i.c.v.) pretreatment with the vasopressin (AVP) V1-receptor antagonist, [d(CH2)5Tyr(Me)]-AVP (0.5 and 2.0 micrograms), dose-dependently inhibited the pressor response to PVN injection of clonidine (10 micrograms), while systemic (i.v.) and local (intra-PVN injection) pretreatments with V1-receptor antagonist (2.0 micrograms) had no effect. These results suggest that the pressor response to microinjection of clonidine into the PVN of conscious rats is mediated by endogenous brain AVP, which is released by activation of alpha 2-adrenoceptors. It is also suggested that the PVN is a possible brain site for the pressor response to i.c.v. injected clonidine.

Adrenergic alpha-Antagonists↗

PubMed1993

Intrathecal GABAB antagonists attenuate the antinociception produced by microinjection of L-glutamate into the ventromedial medulla of the rat.

This study examined whether the antinociception produced by glutaminergic stimulation of neurons in the nucleus raphe magnus (NRM) and nucleus reticularis gigantocellularis pars alpha (NGCp alpha) is mediated by an action of GABA at GABAB receptors in the spinal cord. Rats were pretreated with intrathecal (i.t.) administration of the selective GABAB receptor antagonists phaclofen (100 micrograms) or CGP 35348 (30 micrograms), the serotonin receptor antagonist methysergide (30 micrograms), or vehicle. Fifteen min later, 30 nmol L-glutamate was microinjected into the NRM, NGCp alpha, or sites in the medulla outside these two regions. Microinjection of L-glutamate into the NRM or NGCp alpha in vehicle-pretreated rats significantly increased tail flick latency. This increase was antagonized, but not abolished, by i.t. pretreatment with 30 micrograms CGP 35348 or 100 micrograms phaclofen. Pretreatment with 30 micrograms methysergide completely antagonized the antinociception produced by L-glutamate. Microinjection of L-glutamate at medullary sites outside the NMR or NGCp alpha did not produce antinociception. In an ancillary experiment, the possibility that the ability of methysergide, phaclofen or CGP 35348 to antagonize glutamate-induced antinociception was related to non-specific increases in tail skin temperature was explored. Although phaclofen or methysergide increased tail skin temperature, the magnitude and time course of this increase were not consistent with the antagonism of glutamate-induced antinociception. Moreover, administration of CGP 35348 resulted in a modest decrease in tail skin temperature. Thus, antagonism of glutamate-induced antinociception does not appear to result from non-specific alterations in tail skin temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

Analgesics↗

PubMed1995

The opposing effects of interleukin -1 beta microinjected into the preoptic hypothalamus and the ventromedial hypothalamus on nociceptive behavior in rats.

The effects of microinjections of recombinant human interleukin-1 beta (rhIL-1 beta) into the hypothalamus and neighboring basal forebrain on nociceptive behavior were studied using a hot-plate test in rats. The microinjection of rhIL-1 beta at doses between 5 pg/kg and 50 pg/kg into the medial part of the preoptic area (MPO) reduced the paw-withdrawal latency. The maximal reduction was obtained 30 min after the injection of rhIL-1 beta at 20 pg/kg. RhIL-1 beta (20 pg/kg)-induced hyperalgesia was completely blocked by the simultaneous injection of IL-1 receptor antagonist (IL-1ra, 20 ng/kg), Na salicylate (200 ng/kg) or alpha-melanocyte-stimulating hormone alpha-MSH, 20 ng/kg). The intra-MPO injection of rhIL-1 beta at doses of less than 5 pg/kg or more than 50 pg/kg (up to 2 ng/kg) into the paraventricular nucleus, the lateral hypothalamic area and the septal nucleus had no effect on nociception. The microinjection rhIL-1 beta (20 pg/kg-50 pg/kg) into the ventromedial hypothalamus produced a prolongation of the paw-withdrawal latency. A maximal prolongation was obtained 10 min after the injection of rhIL-1 beta at 50 pg/kg. This reaction was also blocked by the simultaneous injection of IL-1ra (50 ng/kg) and Na salicylate (500 ng/kg). These findings indicate that IL-1 beta in the MPO and the VMH produces hyperalgesia and analgesia, respectively, while, in addition, both effects are mediated by IL-1 receptors and the synthesis of prostaglandins.

Animals↗

PubMed1996

Procaine microinjection into the lower midbrain increases brown fat and body temperatures in anesthetized rats.

A tonic inhibitory mechanism on heat production was studied by microinjecting procaine into various regions of the brain while recording temperature changes of the interscapular brown adipose tissue (IBAT) and rectum in urethane-anesthetized rats at room temperature of 23-25 degrees C. Procaine microinjected bilaterally (10%, 1.0 mu l/site, 1.5 mm to midline) into the midbrain and the upper- to mid-pontine area of the reticular formation increased temperatures of the IBAT and rectum. The highest temperature rise (1.02 +/- 0.11 degrees C for IBAT, 0.64 +/- 0.06 degrees C for rectum) with the shortest onset latency (1.5 +/- 0.3 min for IBAT, 4.6 +/- 1.1 min for rectum) was observed when procaine was injected into the lower midbrain (the area between 6 and 7 mm posterior to the bregma, and 6.5 to 8.5 mm deep from the cortical surface). These regions include the retrorubral field, pedunculopontine tegmental nucleus, and rubrospinal tract. Procaine-induced IBAT and rectal temperature increases were dose-dependent, and reproduced reliably from the same injection site of the same animal. Intravenous indomethacin, a prostaglandin H synthase inhibitor, did not affect procaine-induced temperature rise, and propranolol, a beta-blocker, completely blocked it. These results suggest that microinjected procaine exerts its local anesthetic effect and release a tonic inhibition resulting in a disinhibition-induced temperature increase through the enhanced central sympathetic outflow. They support the hypothesis that a bilateral tonic inhibitory mechanism on heat production exists in the lower midbrain.

Adipose Tissue, Brown↗

PubMed1996

Spinal cholinergic and monoamine receptors mediate the antinociceptive effect of morphine microinjected in the periaqueductal gray on the rat tail, but not the feet.

The antinociceptive effects of morphine (5 micrograms) microinjected into the ventrolateral periaqueductal gray were determined using both the tail flick and the foot withdrawal responses to noxious radiant heating in lightly anesthetized rats. Intrathecal injection of appropriate antagonists was used to determine whether the antinociceptive effects of morphine were mediated by alpha 2-noradrenergic, serotonergic, opioid, or cholinergic muscarinic receptors. The increase in the foot withdrawal response latency produced by microinjection of morphine in the ventrolateral periaqueductal gray was reversed by intrathecal injection of the cholinergic muscarinic receptor antagonist atropine, but was not affected by the alpha 2-adrenoceptor antagonist yohimbine, the serotonergic receptor antagonist methysergide, or the opioid receptor antagonist naloxone. In contrast, the increase in the tail flick response latency produced by morphine was reduced by either yohimbine, methysergide or atropine. These results indicate that microinjection of morphine in the ventrolateral periaqueductal gray inhibits nociceptive responses to noxious heating of the tail by activating descending neuronal systems that are different from those that inhibits the nociceptive responses to noxious heating of the feet. More specifically, serotonergic, muscarinic cholinergic and alpha 2-noradrenergic receptors appear to mediate the antinociception produced by morphine using the tail flick test. In contrast, muscarinic cholinergic, but not monoamine receptors appear to mediate the antinociceptive effects of morphine using the foot withdrawal response.

Animals↗

PubMed1984

Stimulation of DNA synthesis by microinjection of diadenosine 5',5''-P1, P4-tetraphosphate (Ap4A) into Xenopus laevis oocytes.

Ap4A stimulated DNA synthesis when injected into oocytes. The stimulation was dramatically increased when an exogenous template was microinjected. Aphidicolin inhibited the effect of Ap4A, supporting a role of DNA polymerase alpha in this process. No stimulation by Ap4A was observed in microinjected eggs, nor ATP was able to mimic the in vivo effect of Ap4A. Besides microinjected activated DNA, the stimulation by Ap4A of DNA synthesis was also observed with poly dT and poly dT-poly dA as templates, while no effect was observed with poly dA- dT12 -18 and poly dC- dG12 -18. These results support a role of Ap4A in the initiation of DNA synthesis.

Adenine Nucleotides↗

PubMed1989

Egg-induced modifications of the zona pellucida of mouse eggs: effects of microinjected inositol 1,4,5-trisphosphate.

Mouse eggs microinjected with physiological concentrations of inositol 1,4,5-trisphosphate (IP3) do not emit the second polar body, form a pronucleus, or display a fertilization-associated set of changes in the pattern of protein synthesis. IP3-injected eggs, however, display a conversion of the zona pellucida glycoprotein ZP2 to ZP2f. The effect is concentration-dependent with an EC50 (effective concentration, 50%) of 5-10 nM and also occurs in the presence of reduced levels of extracellular calcium. The egg-induced zona pellucida modification is not elicited by several other inositol phosphates that are not able to release calcium from intracellular stores in other systems. Analysis of individual eggs microinjected with IP3 reveals a strong correlation between a reduced binding of sperm to the zona pellucida and the ZP2 to ZP2f conversion. In addition, solubilized zonae pellucidae isolated from IP3-injected eggs possess reduced levels of acrosome reaction-inducing activity. These egg-induced modifications of the zona pellucida--reduced sperm receptor and acrosome reaction-inducing activities and the ZP2 to ZP2f conversion--elicited by microinjected-IP3 are similar to those that occur following fertilization. Results of these experiments suggest that IP3 generated in response to fertilization may play a role in the egg-induced modifications of the zona pellucida that result in the polyspermy block.

Acrosome↗

PubMed1989

Cardiorespiratory effects of inositol hexakisphosphate following microinjections into the nucleus tractus solitarii.

Microinjections of inositol hexakisphosphate (IP6), a metabolite of inositol recently found to occur in high concentrations in the brainstem, were made into the caudal portion of the nucleus tractus solitarii (NTS) of spontaneously breathing rats and cardiorespiratory parameters recorded for a 30 min test period. Microinjections of IP6, in the dose range of 100-500 pmol/rat, produced significant dose-related reductions in mean arterial blood pressure and respiratory rate. The onset for hypotensive action and respiratory depression following microinjections of IP6 was very rapid and a transient apnea could be elicited at the higher doses. Moreover, the sodium and calcium salts of IP6 were relatively equipotent in depressing cardiorespiratory parameters, with the exception of heart rate wherein the sodium salt elicited a much more pronounced bradycardia. These results confirm and extend the findings of a previous study suggesting that IP6 and closely related metabolites may act on extracellular receptors. Taken together, these data provide further support to the notion that inositol lipid signalling pathways may generate both intracellular and extracellular signals in the brain.

Animals↗

PubMed1986

Protein phosphorylation and oocyte maturation. I. Induction of starfish oocyte maturation by intracellular microinjection of a phosphatase inhibitor, alpha-naphthylphosphate.

Oocyte maturation (meiosis re-initiation) in starfish is induced by the natural hormone 1-methyladenine (1-MeAde). Following hormonal stimulation of the oocyte, an intracellular Maturation Promoting Factor (MPF) appears in the cytoplasm which triggers nuclear envelope breakdown and maturation divisions. alpha-Naphthylphosphate (alpha-NP), a widely used phosphatase inhibitor/substrate, was found to induce oocyte maturation when microinjected intracellularly (50% maturation of 3.5 mM; 100% above 6mM, final intracellular concentration) into oocytes of Marthasterias and Asterias but not of Astropecten. As 1-MeAde, alpha-NP triggers a complete maturation, i.e. germinal vesicle breakdown, extrusion of the two polar bodies and formation of the female pronucleus. The kinetics of alpha-NP-induced maturation (35-45 min) is, however, longer than the kinetics of 1-MeAde-induced maturation (18-20 min). The addition of alpha-NP externally to oocytes does not trigger maturation. Among several reported phosphatase inhibitors, including two natural protein phosphatase inhibitors and several products structurally related to alpha-NP, only alpha-NP was found capable of inducing maturation when microinjection into oocytes. alpha-NP triggers the appearance of MPF activity in the cytoplasm of oocytes into which it has been injected. Although alpha-NP-induced maturation is insensitive to inhibitors whose action is known to be restricted to the hormone-dependent period (such as the protease inhibitor leupeptin), it is blocked by inhibitors of MPF action (such as nicotinamide and lithium). Finally it was found that alpha-NP-induced maturation is inhibited by simultaneous microinjection of protein phosphatase-2A; also, alpha-NP, classically used as an inhibitor of acid and alkaline phosphatases, is able to inhibit protein phosphatases, is able to inhibit protein phosphatases 1 and 2 A. The addition of alpha-NP to oocytes increases the level of phosphorylated proteins. These results constitute direct evidence that an elevated level of phosphorylated proteins is sufficient to trigger MPF activity and to induce maturation.

Alkaline Phosphatase↗

PubMed1986

Protein phosphorylation and oocyte maturation. II. Inhibition of starfish oocyte maturation by intracellular microinjection of protein phosphatases 1 and 2A and alkaline phosphatase.

Oocyte maturation (meiosis re-initiation) in starfish is induced by the natural hormone 1-methyladenine (1-MeAde). Following hormonal stimulation of the oocyte, an intracellular Maturation Promoting Factor (MPF) appears in the cytoplasm which triggers nuclear envelope breakdown and maturation divisions. Microinjection of pure preparations of the catalytic subunits of protein phosphatases 1 and 2A inhibits 1-MeAde-induced maturation in a dose-dependent manner. Calmodulin-dependent protein phosphatase 2B is inefficient. Maturation induced by mimetics of 1-MeAde, such as dithiothreitol (DTT), methylglyoxal-bis(guanylhydrazone) (MGBG), 8-hydroxyeicosatetraenoic acid (8 HETE) and arachidonic acid (AA) is also inhibited by these protein phosphatases. In all cases inhibition can be reversed by increasing the concentration of 1-Me-Ade or of mimetic. Alkaline phosphatase also inhibits maturation in a dose-dependent way and in a reversible manner. Microinjection of protein phosphatase is still effective when preformed long after the end of the hormone-dependent period, and can even be effective a few minutes before the breakdown of the nuclear envelope. No detectable MPF activity is found in 1-MeAde-treated phosphatase-injected oocytes. However, microinjection of phosphatase 2A simultaneously with MPF (obtained from 1-MeAde-treated donors) does not result in inhibition. These results constitute direct evidence for the necessity of an elevated level of phosphorylated proteins for MPF activity and maturation. The mode of action of 1-MeAde in inducing starfish oocyte maturation is discussed in relation to protein phosphorylation.

Adenine↗