Leucokinin and diuretic hormone immunoreactivity of neurons in the tobacco hornworm,Manduca sexta,and co-localization of this immunoreactivity in lateral neurosecretory cells of abdominal ganglia

Because leucokinins stimulate diuresis in some insects, we wished to identify the neurosecretory cells in Manduca sexta that might be a source of leucokinin-like neurohormones. Immunostaining was done at various stages of development, using an antiserum to leucokinin IV. Bilateral pairs of neurosecretory cells in abdominal ganglia 3–7 of larvae and adults are immunoreactive; these cells project via the ipsilateral ventral nerves to the neurohemal transverse nerves. The immunoreactivity and size of these lateral cells greatly increases in the pharate adult, and this change appears to be related to a period of intensive diuresis occurring a few days before adult eclosion. Relationships of these neurons to cells that are immunoreactive to a M. sexta diuretic hormone were also investigated. Diuretic hormone and leucokinin immunoreactivity are co-localized in the lateral neurosecretory cells and their neurohemal projections. A median pair of leucokinin-immunoreactive, and a lateral pair of diuretic hormone-immunoreactive neurons in the larval terminal abdominal ganglion project to neurohemal release sites within the cryptonephridium. The immunoreactivity of these cells is lost as the cryptonephridium is eliminated during metamorphosis. This loss appears to be related to the change from the larval to adult pattern of diuresis.     

Dynamics and metamorphosis of an identifiable peptidergic neuron in an insect

Eclosion hormone (EH) is a 7000 Da peptide that triggers ecdysis behavior in insects. In the moth, Manduca sexta, EH is found in two pairs of ventromedial (VM) cells in the brain which send their axons down the ventral nerve cord to a neurohemal site in the proctodeal nerve in the larva and pupa. During adult development, these cells send axon collaterals to the corpora cardiaca where they form a new release site used for adult eclosion. Studies of bioassayable peptide during the 5th larval instar and the larval-pupal transformation revealed that after depletion at ecdysis, the VM cells showed a transient increase in EH found in their cell bodies and axons. By contrast, their terminals in the proctodeal nerve showed a gradual accumulation of peptide followed by a release of over 90% of the stored material at pupal ecdysis. In situ hybridization analysis on whole mounts of the brains showed that the VM cells always contained EH mRNA with increased accumulation during the larval and pupal molting periods with a slight decline just before ecdysis. High levels of EH mRNA were found in brains of diapausing pupae. During the first two-thirds of adult development, mRNA accumulated to high levels, then slowly declined until ecdysis. EH mRNA levels up to 3 days after adult eclosion. At no time was EH mRNA found in the lateral neurosecretory cell cluster previously reported to produce EH for adult eclosion. 1994 John Wiley & Sons, Inc.     

Ca2+-induced Ca2+ release supports the relay mode of activity in thalamocortical cells

Ca2+ ions play an important role during rhythmic bursting of thalamocortical neurons within sleep. The function of Ca2+ during the tonic relay mode of these neurons during wakefulness is less clear. Here, we report that tonic activity in thalamocortical cells results in an increase in the intracellular Ca2+ concentration and subsequent release of Ca2+ from intracellular stores mediated via ryanodine receptors (RyRs). Blockade of Ca2+ release shifted the regular firing of single action potentials toward the generation of spike clusters. Regular spike firing and intracellular Ca2+ release thus appear to be functionally coupled in a positive feedback manner, thereby supporting the relay mode of thalamocortical cells during wakefulness. Regulatory influences may be coupled to this system via the cyclic ADP ribose pathway.     

Aminergic neuron systems of lobsters: morphology and electrophysiology of octopamine-containing neurosecretory cells

In the American lobster (Homarus americanus) the biogenic amines serotonin and octopamine appear to play important and opposite roles in the regulation of aggressive behavior, in the establishment and/or maintenance of dominant and subordinate behavioral states and in the modulation of the associated postural stances and escape responses. The octopamine-containing neurosecretory neurons in the thoracic regions of the lobster ventral nerve cord fall into two morphological subgroups, the root octopamine cells, a classical neurohemal group with release regions along second thoracic roots, and the claw octopamine cells, a group that selectively innervates the claws. Cells of both subgroups have additional sets of endings within neuropil regions of ganglia of the ventral nerve cord. Octopamine neurosecretory neurons generally are silent, but when spontaneously active or when activated, they show large overshooting action potentials with prominent after-hyperpolarizations. Autoinhibition after high-frequency firing, which is also seen in other crustacean neurosecretory cells, is readily apparent in these cells. The cells show no spontaneous synaptic activity, but appear to be excited by a unitary source. Stimulation of lateral or medial giant axons, which excite serotonergic cells yielded no response in octopaminergic neurosecretory cells and no evidence for direct interactions between pairs of octopamine neurons, or between the octopaminergic and the serotonergic sets of neurosecretory neurons was found.     

Three-dimensional architecture of identified cerebral neurosecretory cells in an insect

The organization of identified neurosecretory cell groups in the larval brain of the tobacco hornworm, Manduca sexta, was investigated immunocytologically. Computer-assisted three-dimensional reconstruction was used to examine the architecture of the neurosecretory cell groups. The group III lateral neurosecretory cells (L-NSC-III) which produce the prothoracicotropic hormone are located dorsolaterally in the protocerebrum and extend axons medially that decussate to the contralateral lobe prior to exiting the brain through the nervi corporis cardiaci I + II. The group IIa2 medial neurosecretory cells (M-NSC IIa2) are located anteriorly in the medial dorsal protocerebrum. The axons of these cells also exit the brain via the contralateral nervi corporis cardiaci I + II. However, their axons traverse a different pathway through the brain from that of the L-NSC III axons. Each of the cell groups possesses elaborate dendrites with terminal varicosities. The dendrites can be classified into specific fields based upon their location and projection pattern within the brain. The dendrites for these two neurosecretory cell groups overlap in specific regions of the protocerebral neuropil. After the axons of these neurosecretory cells exit the brain through the retrocerebral nerve, they innervate the corpus allatum where they arborize to form neurohemal terminals in strikingly different patterns. The L-NSC III penetrate throughout the glandular structure and the M-NSC IIa2 terminals are restricted to the external sheath. A third group of cerebral neurosecretory cells, the ventromedial neurons (VM) which stain with the monoclonal antibody to prothoracicotropic hormone in Manduca, are located anteriorly in the medial region of the brain. The axons of these cells do not exit the brain to the retrocerebral complex, but rather pass through the circumesophageal connectives and ventral nerve cord. These neurons appear to be the same VM neurons that produce eclosion hormone. One dendritic field of the L-NSC III terminates in close apposition to the VM neurons. The distinct morphologies of these neurosecretory cell groups in relation to other cell groups and the distribution of neuropeptides within the neurons suggest that insect neurosecretory cells, like their vertebrate counterparts, may have multiple regulatory roles.     

Development of the gin trap reflex inManduca sexta: a comparison of larval and pupal motor responses

1. Responses of motor neurons in larvae and pupae of Manduca sexta to stimulation of tactile sensory neurons were measured in both semi-intact, and isolated nerve cord preparations. These motor neurons innervate abdominal intersegmental muscles which are involved in the production of a general flexion reflex in the larva, and the closure reflex of the pupal gin traps. 2. Larval motor neurons respond to stimulation of sensory neurons innervating abdominal mechanosensory hairs with prolonged, tonic excitation ipsilaterally, and either weak excitation or inhibition contralaterally (Figs. 4A, 6). 3. Pupae respond to tactile stimulation of mechanosensory hairs within the gin traps with a rapid closure reflex. Motor neurons which innervate muscles ipsilateral to the stimulus exhibit a large depolarization, high frequency firing, and abrupt termination (Figs. 2, 4B). Generally, contralateral motor neurons fire antiphasically to the ipsilateral motor neurons, producing a characteristic triphasic firing pattern (Figs. 7, 8) which is not seen in the larva. 4. Pupal motor neurons can also respond to sensory stimulation with other types of patterns, including rotational responses (Fig. 3A), gin trap opening reflexes (Fig. 3B), and 'flip-flop' responses (Fig. 9). 5. Pupal motor neurons, like larval motor neurons, do not show oscillatory responses to tonic current injection, nor do motor neurons of either stage appear to interact synaptically with one another. Most pupal motor neurons also exhibit i-V properties similar to those of larval motor neurons (Table 1; Fig. 10). Some pupal motor neurons, however, show a marked non-linear response to depolarizing current injection (Fig. 11).     

Firing activities of neurosecretory cells producing diapause hormone and its related peptides in the female silkmoth,Bombyx mori. I. labial cells

There are three known clusters of neurosecretory cells expressing a gene encoding diapause hormone (DH) and four related peptides in the suboesophageal ganglion (SOG) of Bombyx mori. Long-term chronic recordings were made from the axonal tract (NCC-3) of a pair of cells localized in the labial (posterior) neuromere of SOG during pupal-adult development. There was a significant difference in firing activity patterns of the labial neurosecretory cells between diapause-egg and non-diapause-egg producers: labial cells in the former were active throughout pupal-adult development, whereas the same cells in the latter usually maintained an inactive state until the last quarter of pupal-adult development, a time at which a secretion of DH seems to be too late to act on the developing ovary for the induction of diapausing eggs. This observation strongly supports the notion that labial cells release DH and are responsible for determination of embryonic diapause in the silkmoth.     

Immunohistochemical investigation of urotensins in the caudal spinal cord of four species of elasmobranchs and the lamprey,Lampetra japonica

Summary In the four species of elasmobranchs examined (Triakis scyllia, Heterodontus japonicus, Scyliorhinus torazame, Dasyatis akajei), all identifiable caudal neurosecretory cells and their corresponding neurohemal areas showed urotensin II (UII)-immunoreactivity with varied intensity. To localize urotensin I (UI) in the caudal neurosecretory system of the dogfish, Triakis scyllia, h-CRF (1–20) antiserum that cross-reacts with UI was used in place of UI antiserum. CRF/UI-immunoreactivity was demonstrated in the neurosecretory cells and neurohemal areas. A considerable number of neurons showed both UII- and CRF/UI-immunoreactivities, suggesting that UII and UI are produced in the same neurosecretory cells. However, some neurons exhibited UII-immunoreactivity, but no CRF/UI-immunoreactivity. Cells immunoreactive only to CRF antiserum were not detected. At least two populations of neurons exist in the dogfish caudal neurosecretory system: (i) cells immunoreactive for both CRF/UI and UII, and (ii) cells immunoreactive for UII. The dorsal cells of the lamprey, Lampetra japonica, did not react with either UII or CRF antiserum.     

Estrus synchronization in beef cows: comparison between GnRH+PGF2alpha+GnRH and PRID+PGF2alpha+eCG

The aim of this study was to compare two protocols for estrus synchronization in suckled beef cows over a 2 years period. The population studied consisted of 172 Charolais and 168 Limousin cows from 12 and 14 beef herds, respectively. In each herd, cows were allotted to groups according to parity, body condition score and calving difficulty. Cows in Group 1 (n=174) received PRID on Day-8 with estradiol benzoate (10mg, vaginal capsule), dinoprost on Day-4 (25mg i.m.), eCG on Day 2 (500 IU i.m.). The PRID was removed on Day-2 and cows were inseminated on Day 0, 56 h after PRID was removed. Cows in Group 2 (n=166) received GnRH on Day-10 (100 microg i.m.), dinoprost on Day-3 (25mg i.m.) and GnRH on Day-1 (100 microg i.m.), and were inseminated on Day 0, 16-24h after the last GnRH treatment. Plasma progesterone concentrations were measured to determine cyclicity prior to treatment (Days-20 and -10), to confirm the occurrence of ovulation (Days 0 and 10) and to determine the apparent early pregnancy rate (Days 0, 10 and 24). Pregnancy diagnosis was performed by ultrasonography between Days 35 and 45. The effects of various factors on ovulation, apparent early pregnancy and pregnancy rates were studied using logistic mixed models. There was no significant difference between Groups 1 and 2, respectively, for the cyclicity rate before treatment (80.5% versus 80.1%), for apparent pregnancy rate on Day 24 (62.1% versus 54.8%, P=0.09) and for pregnancy rate on Days 35-45 (53.8% versus 46.3%, P=0.16). Ovulation rate was higher (P<0.01) in Group 1 (90.8%) than in Group 2 (77.1%) and was affected by cyclicity prior to treatment in Group 2 but not in Group 1 (Group 1: 88.2% in anestrous cows versus 91.4% in cyclic cows; Group 2: 45.5% in anestrous cows versus 85.0% in cyclic cows, P interaction=0.05). Apparent pregnancy rates on Day 24 were influenced by the year of study (52.4% versus 68.8%, OR=2.12, P<0.01) and by the cyclicity before treatment (anestrous cows 46.3% versus cyclic cows 61.5%, OR=1.86, P<0.05). Pregnancy rates at 35-45 days were influenced by the year of study (44.2% versus 59.8%, OR=1.92, P<0.01). In conclusion, although pregnancy rates were similar for the two treatments, the combination of GnRH+PGF2alpha+GnRH in suckled beef cows induced a lower rate of ovulation than treatment with PRID+PGF2alpha, particularly in anestrous cows.     

Development of the caudal neurosecretory system of the nile tilapia Oreochromis niloticus: an immunohistochemical and electron microscopic study

The development of the caudal neurosecretory system (CNSS) of the Nile tilapia, Oreochromis niloticus, has been investigated by means of UI/oCRF (urotensin I/ovine corticotropin-releasing factor) immunohistochemistry and transmission electron microscopy. UI-like immunoreactive perikarya and fibers are first detected in the caudal spinal cord of larval fish about 4 days after hatching (stage 21). In the region of the future urophysis two bundles of strongly immunoreactive neurosecretory fibers are observed. At this stage, neurosecretory axons terminate on the meninx sheath of the spinal cord with immature neurosecretory terminals. The histogenesis of the urophysis begins at stage 24. The future neurohemal organ consists of a small ventral swelling of the spinal cord, which is associated with dilated vessels. At this stage, neurosecretory axons terminate on the basal lamina of the ingrowing blood vessels. Further development occurs by means of progressive branching of vessels and the concomitant increase in the number of neurosecretory terminals. In the caudal spinal cord, immunoreactive neurons also increase in number and progressively differentiate morphologically. Typical features of the mature CNSS are recognizable in 4-month-old juveniles. Data suggest that in tilapia both the synthesis and the release of urophysial hormones begin before morphogenesis of the neurohemal organ takes place.     

Comparison of in-vitro production of progestagens by the corpora lutea of early pregnancy of the rat and hamster

Immature rats and adult hamsters were killed on Days 2, 4 or 8 of pregnancy (Day 1 = sperm positive vaginal smear). Dispersed luteal cells (5 X 10(4) cells) were incubated for 2 h in the absence or presence of graded doses of ovine LH. In the absence of LH, incubation of rat luteal cells compared to hamster cells produced about 3-6-fold as much progesterone, 26-66 times as much 20 alpha-dihydroprogesterone and about the same amounts of 17 alpha-hydroxyprogesterone. For the rat, 1 ng LH was the minimal dose which stimulated synthesis of progesterone and 17 alpha-hydroxyprogesterone by luteal cells on Days 2 and 4 whereas 10 ng LH stimulated maximal production of progesterone by Day-8 luteal cells. As pregnancy progressed from Day 2 to Day 8, there was an inverse relationship between the levels of progesterone and 20 alpha-dihydroprogesterone accumulated by rat luteal cells. For the hamster, 1 ng LH significantly stimulated accumulation of progesterone and 17 alpha-hydroxyprogesterone by Day-2 luteal cells but not by Day-4 or Day-8 cells. Hamster luteal cells on Day 4 produced the highest levels of progesterone in response to 10 or 100 ng LH, with a maximal rate of accumulation by Day-8 cells with 10 ng LH.     

Effect of variation in embryo stage on the establishment of pregnancy, and embryo survival and growth in ewes with two embryos

Embryos at different stages of development were transferred to recipient ewes on Day 6 to investigate the effect of variation in stage of development on embryo survival and growth. Three groups of ewes received 2 embryos that were at the same stage of development, Day 4, Day 6 or Day 8. A fourth group received 1 Day-4 and 1 Day-8 embryo. At autopsy on recipient Day 34 there were no significant differences in embryo survival (Day 4, 34%; Day 6, 50%; Day 8, 46%; and Day 4 and 8, 48%). Fetuses developing from Day-8 embryos were heavier than others (Day 4, 1.10 +/- 0.06 g; Day 6, 1.15 +/- 0.06 g; Day 8, 1.41 +/- 0.08 g; P less than 0.05). In Group 4 neither survival nor growth of embryos was significantly affected by the presence of an embryo at a different stage of development. The ability of the uterus to stimulate development of a relatively retarded embryo is confirmed. Apparently the uterus has less effect in slowing the development of advanced embryos.     

The neurosecretory system of the adult Melanogryllus desertus pall. (Orthoptera,Gryllidae)

Summary The cerebral neurohemal area of Melanogryllus desertus is located posteriorly among the neurons of nervus corporis cardiaci I (NCCI) on the ventral median surface of the protocerebrum where axons penetrate the neural lamella and terminate on its outer surface. Numerous neurosecretory fibers containing three different types of granule occur within and on the outer surface of the neural lamella.The release of neurosecretory granules is accomplished by exocytosis and the formation of synaptoids. It can also take place as a mass release of granules into the stroma.     

Retrograde Gbb signaling through the Bmp type 2 receptor wishful thinking regulates systemic FMRFa expression in Drosophila

Amidated neuropeptides of the FMRFamide class regulate numerous physiological processes including synaptic efficacy at the Drosophila neuromuscular junction (NMJ). We demonstrate here that mutations in wishful thinking (wit) a gene encoding a Drosophila Bmp type 2 receptor that is required for proper neurotransmitter release at the neuromuscular junction, also eliminates expression of FMRFa in that subset of neuroendocrine cells (Tv neurons) which provide the systemic supply of FMRFa peptides. We show that Gbb, a Bmp ligand expressed in the neurohemal organ provides a retrograde signal that helps specify the peptidergic phenotype of the Tv neurons. Finally, we show that supplying FMRFa in neurosecretory cells partially rescues the wit lethal phenotype without rescuing the primary morphological or electrophysiological defects of wit mutants. We propose that Wit and Gbb globally regulate NMJ function by controlling both the growth and transmitter release properties of the synapse as well as the expression of systemic modulators of NMJ synaptic activity.     

Isolation of an eclosion hormone gene from the cotton bollworm, Helicoverpa armigera: temporal and spatial distribution of transcripts

The cDNA encoding eclosion hormone (EH), which plays an integral role in triggering ecdysis behavior at the end of each molt, was cloned from the cotton bollworm, Helicoverpa armigera (Har) (Lepidoptera: Noctuidae). The EH polyprotein precursor contains a 26-amino acid signal peptide and a single 62-amino acid mature EH. Compared the mature Har-EH with other known EHs, it shows 94%, 84%, and 59% identities to Manduca sexta, Bombyx mori, and Drosophila melanogaster, respectively. Har-EH mRNA is expressed only in the brain by Northern blot and RT-PCR, but not in other tissues. By in situ hybridization and immunocytochemistry, both Har-EH mRNA and protein are localized in two pairs of neurosecretory cells of the brain. Prior to a molt, expression level of Har-EH gene reaches the highest point, and then drops after molt. EH release is detected both centrally, within the ganglia, and peripherally, into the hemolymph. A peak of the EH titer in hemolymph measured by ELISA presents at ecdysis. These results are consistent with the biological function of Har-EH associated with ecdysis. Furthermore, Har-EH gene is expressed throughout all of the developmental stages examined, implicating that the EH gene may possess other biological functions in post-embryonic development other than triggering ecdysis behavior.     

Evidence for brain-derived neurotrophic factor-like neuropeptide in brain of the silk moth Bombyx mori during postembryonic periods

Brain-derived neurotrophic factor-like neuropeptide is produced in the brain of the silk moth, Bombyx mori. Immunocytochemical studies of brain and retrocerebral complex of larvae, prepupae, pupae and adults showed that four pairs of median neurosecretory cells and six pairs of lateral neurosecretory cells which had different immunoreactivities to BDNF peptide. Day-1 adult brains showed no evidence of neurons stained by anti-BDNF antibodies. Those reactivities, which were much stronger in median cells than in lateral cells, were the weakest in an earliest larval stage and a latest pupal stage but the strongest in late larval stage. Median neurosecretory cells projected their axons into the contralateral corpora allata by decussation in the median region, nerve corpora cardiaca (NCC) I, and nerve corpora allata (NCA) I, whereas lateral neurosecretory cells extended their axons to the ipsilateral corpora allata via NCC II and NCA I.     

Diversity of neurohormonal release sites in insects: Neurohemal areas associated with peripheral neurosecretory cells in Periplaneta americana L. (Dictyoptera: Blattidae) and Locusta migratoria R. & F. (Orthoptera : Locustidae)

Neurohemal areas are located on the distal region of the transverse nerve and on the link nerve of the cockroach, Periplaneta americana (Dictyoptera : Blattidae) and the locust, Locusta migratoria (Orthoptera : Locustidae). They constitute the sites at which the peripheral neurosecretory cells on these nerves discharge their products. Histological and ultrastructural studies suggest that, at least in Periplaneta, these areas also serve to release neurosecretory products different from those found in the perisympathetic organs (POs). These products come from the ganglion cells, some of them via the transverse nerve, and others, via the somatic nerves. The existence of these neurohemal areas broadens the problem of the release zones and shows that the POs are not the only neurohemal structures associated with ventral ganglion cells. An attempt is made to explain the reason for the existence of these different release sites, as well as the role of the peripheral cells.     

昆虫蜕皮行为的生理生化和分子生物学研究进展

羽化激素与蜕皮触发激素诱发昆虫蜕皮行为及蜕皮末期的其它生理变化。羽化激素在一些特定的脑神经分泌细胞中合成,在蜕皮激素的调控下,释放到中枢神经系统和血淋巴中。蜕皮触发激素是由Inka细胞分泌的,直接作用于中枢神经系统,触发前蜕皮和蜕皮行为。越来越多的证据表明羽化激素可能存在于所有的昆虫中,并作为一种调节蜕皮的一般性激素机制。     

Homeostatic regulation mechanism of spontaneous firing determines glutamate responsiveness in the midbrain dopamine neurons

Autonomous tonic firing of the midbrain dopamine neuron is essential for maintenance of ambient dopamine level in the brain, in which intracellular Ca²⁺ concentration ([Ca²⁺]c) plays a complex but pivotal role. However, little is known about Ca²⁺ signals by which dopamine neurons maintain an optimum spontaneous firing rate. In the midbrain dopamine neurons, we here show that spontaneous firing evoked [Ca²⁺]c changes in a phasic manner in the dendritic region but a tonic manner in the soma. Tonic levels of somatic [Ca²⁺]c strictly tallied with spontaneous firing rates. However, manipulatory raising or lowering of [Ca²⁺]c with caged compounds from the resting firing state proportionally suppressed or raised spontaneous firing rate, respectively, suggesting presence of the homeostatic regulation mechanism for spontaneous firing rate via tonic [Ca²⁺]c changes of the soma. More importantly, abolition of this homeostatic regulation mechanism significantly exaggerated the responses of tonic firings and high-frequency phasic discharges to glutamate. Therefore, we conclude that this Ca²⁺-dependent homeostatic regulation mechanism is responsible for not only maintaining optimum rate of spontaneous firing, but also proper responses to glutamate. Perturbation of this mechanism could cause dopamine neurons to be more vulnerable to glutamate and Ca²⁺ toxicities.     

Signal transduction in eclosion hormone-induced secretion of ecdysis-triggering hormone

Inka cells of insect epitracheal glands (EGs) secrete preecdysis and ecdysis-triggering hormones (PETH and ETH) at the end of each developmental stage. Both peptides act in the central nervous system to evoke the ecdysis behavioral sequence, a stereotype behavior during which old cuticle is shed. Secretion of ETH is stimulated by a brain neuropeptide, eclosion hormone (EH). EH evokes accumulation of cGMP followed by release of ETH from Inka cells, and exogenous cGMP evokes secretion of ETH. The secretory responses to EH and cGMP are inhibited by the broad-spectrum kinase inhibitor staurosporine, and the response to EH is potentiated by the phosphatase inhibitor calyculin A. Staurosporine did not inhibit EH-evoked accumulation of cGMP. Changes in cytoplasmic Ca2+ in Inka cells during EH signaling were monitored via fluorescence ratioing with fura-2-loaded EGs. Cytoplasmic Ca2+ increases within 30-120 s after addition of EH to EGs, and it remains elevated for at least 10 min, corresponding with the time course of secretion. Secretion is increased in dose-dependent manner by the Ca2+-ATPase inhibitor thapsigargin, a treatment that does not elevate glandular cGMP above basal levels. The secretory response to EH is partially inhibited in glands loaded with EGTA, while cGMP levels are unaffected. These findings suggest that EH activates second messenger cascades leading to cGMP accumulation and Ca2+ mobilization and/or influx and that both pathways are required for a full secretory response. cGMP activates a staurosporine-inhibitable protein kinase. We propose that Ca2+ acts via a parallel cascade with a time course that is similar to that for cGMP activation of a cGMP-dependent protein kinase.