Nathan J. Tublitz

Amino acid sequence of CAP2b, an insect cardioacceleratory peptide from the tobacco hawkmoth Manduca sexta

The primary structure of a novel insect neuropeptide, Cardioacceleratory Peptide 2b (CAP2b), from the tobacco hawkmoth Manduca sexta has been established using a combination of mass spectroscopy, Edman degradation microsequencing, amino acid analysis, and biological assays. The sequence of CAP2b, pyroGlu‐Leu‐Tyr‐Ala‐Phe‐Pro‐Arg‐Val‐amide, has a molecular weight of 974.6 and is blocked at both the amino and car☐yl ends. Examination of several national computer protein data bases failed to reveal other peptides or proteins with any sequence homology to CAP2b indicating that this is likely to be a novel insect neuropeptide. This peptide may be a general activator of insect viscera since it causes an increase in heart rate in Manduca and in Drosophila, and has also been implicated in the regulation of fluid secretion by the Malphigian tubules of Drosophila.

Primary structure of a cardioactive neuropeptide from the tobacco hawkmoth, Manduca sexta

The amino acid sequence of the first of a family of insect cardioregulatory peptides from the tobacco hawkmoth, Manduca sexta, has been determined using a combination of Edman degradation microsequencing and mass spectroscopy. This peptide contains 9 amino acid residues and an observed mass for the monoisotopic protonated molecule of 956.4 Da. There are two cysteines at positions 3 and 9 forming a disulfide bridge and the carboxyl‐terminus is amidated. The structure of this peptide, Pro‐Phe‐Cys‐Asn‐Ala‐Phe‐Thr‐Gly‐Cys‐NH2, is identical to a peptide recently isolated from crabs called crustacean cardioactive peptide (CCAP) and we propose that this peptide be named Manduca CCAP.

From behavior to molecules: an integrated approach to the study of neuropeptides

Despite extensive information on many aspects of peptide neurobiology, the links between the behavioral effects of neuropeptides and their actions at the cellular and molecular levels are not fully understood. A pair of insect neuropeptides, the cardioacceleratory peptides (CAPs) of the tobacco hawkmoth Manduca sexta, provide an opportunity to elucidate these links. The CAPs are involved in the modulation of four distinct types of behavior during the life cycle of this moth. Functional differences at these four developmental periods can be explained by stage-specific changes in target sensitivity and the distribution of the CAP-containing neurons, including a set of peptidergic neurons that alter their transmitter phenotype postembryonically. Studies show that inositol 1, 4, 5-trisphosphate (IP3), linked to intracellular Ca2+, mediates the response of the cells to the CAPs. This preparation thus provides additional insights into the mechanisms underlying the action of multifunctional neuropeptides.

Quantitative staging of embryonic development of the tobacco hawkmoth,Manduca sexta

SummaryA complete timetable of embryonic development of the tobacco hawkmoth,Manduca sexta (Lepidoptera: Sphingidae), is presented. Using living embryos, 20 developmental stages from oviposition to hatching are described with respect to their morphological and physiological maturation. This staging series provides a simple method to identify the stage ofManduca development during all phases of embryogenesis.

Peptidergic regulation of behavior: an identified neuron approach

Abstract The various physiological and behavioral roles of neuropeptides have caused a general revision in our understanding of the importance of these neurochemical messengers in the control of CNS function. Yet little is known about the functional regulation of neuropeptides at the level of the individual peptidergic cell. Here we discuss three inter-twined peptidergic systems that are involved in the control of several related behaviors in the tobacco hawkmoth, Manduca sexta. We present experiments that led to the identification of the individual peptide-containing neurons and discuss some of the mechanisms that regulate peptide release for each peptidergic system.

Neurokinin-1 receptor desensitization to consecutive microdialysis infusions of substance P in human skin

The neuropeptide substance P is known to be localized in nerve terminals in human skin and substance P‐induced vasodilatation is believed to be partially dependent on nitric oxide (NO) and H1 histamine receptor activation. Unlike other neuropeptides investigated in human skin, substance P‐induced vasodilatation has been shown to decline during continuous infusion, possibly suggestive of an internalization of neurokinin‐1 (NK1) receptors, which are highly specific to substance P. However, questions remain regarding these mechanisms in human skin. Fifteen subjects participated in this series of studies designed to investigate the effect of consecutive infusions and possible mechanisms of substance P‐induced vasodilatation in human skin. Two concentrations of substance P (10 μm and 20 μm) were tested via intradermal microdialysis in two groups of subjects. Site 1 served as a control and received substance P only. Site 2 received substance P combined with 10 mm l‐NAME to inhibit NO synthase. Site 3 received substance P combined with 500 μm pyrilamine, an H1 receptor antagonist. Site 4 received substance P combined with 10 mm l‐NAME plus 500 μm pyrilamine. Red blood cell (RBC) flux was measured via laser‐Doppler flowmetry to provide an index of skin blood flow. Cutaneous vascular conductance was calculated as RBC flux/mean arterial pressure and was normalized to maximal vasodilatation via 28 mm sodium nitroprusside. Substance P was perfused through each microdialysis fibre at a rate of 4 μl min−1 for 15 min. The subsequent increase in skin blood flow was allowed to return to baseline (∼45–60 min) and a stable 5 min plateau was used as a new baseline (post‐infusion baseline). A second dose of substance P was then delivered to the skin and skin blood flow was monitored for 45–60 min. Substance P produced a dose‐dependent increase in skin blood flow with the concentrations of substance P tested, which was significantly attenuated in the presence of l‐NAME and the combination of l‐NAME plus pyrilamine. However, substance P‐induced vasodilatation was unaffected in the presence of pyrilamine. There was no significant difference between the l‐NAME‐only sites and the l‐NAME plus pyrilamine sites. Importantly, the second dose of substance P did not produce a significant increase in skin blood flow compared to the initial baseline or the post‐infusion baseline. These data suggest substance P‐induced vasodilatation delivered via microdialysis contains an NO component but does not contain an H1 receptor activation component at the doses tested. Additionally, these data provide evidence for NK1 receptor desensitization as there was no observable increase in skin blood flow following a second administration of substance P. This may provide a useful model for studying the role of substance P in the control of skin blood flow in humans.

Sequence and expression of the CAPA/CAP2b gene in the tobacco hawkmoth, Manduca sexta.

SUMMARY The gene coding for cardioacceleratory peptide 2b (CAP2b; pELYAFPRV) has been isolated and sequenced from the moth Manduca sexta (GenBank accession #AY649544). Because of its significant homology to the CAPA gene in Drosophila melanogaster, this gene is called the Manduca CAPA gene. The Manduca CAPA gene is 958 nucleotides long with 29 untranslated nucleotides from the beginning of the sequence to the putative start initiation site. The CAPA gene has a single open reading frame, 441 nucleotides long, that codes for a predicted precursor protein of 147 amino acids. The predicted prepropeptide encodes a single copy of each of three deduced propeptides, a CAP2b propeptide, with a Q substituted for an E at the N-terminus (QLYAFPRVa), and two novel CAP2b-related propeptides (DGVLNLYPFPRVa and TEGPGMWFGPRLa). To reduce confusion and to adopt a more standardized nomenclature, we rename pELYAFPRVa as Mas-CAPA-1 and assign the names of Mas-CAPA-2 to DGVLNLYPFPRVa and Mas-PK-1 (Pyrokinin-1) to TEGPGMWFGPRLa. The spatial and temporal expression pattern of the CAPA gene in the Manduca central nervous system (CNS) was determined in all major post-embryonic stages using in situ hybridization techniques. The CAPA gene is expressed in a total of 27 pairs of neurons in the post-embryonic Manduca CNS. A total of 16 pairs of cells is observed in the brain, two pairs in the sub-esophageal ganglion (SEG), one pair in the third thoracic ganglion (T3), one pair in each unfused abdominal ganglion (A1–A6) and two pairs in the fused terminal ganglion. The mRNA from the CAPA gene is present in nearly every ganglion in each post-embryonic stage. The number of cells expressing the CAPA gene varies during post-embryonic life, starting at 54 cells in first-instar larvae and declining to a minimum of 14 cells midway through adult development.

Roles of glutamate and FMRFamide-related peptides at the chromatophore neuromuscular junction in the cuttlefish, Sepia officinalis

Body patterning behavior, the expression of highly intricate patterns, is ubiquitous among all unshelled cephalopods. These body patterns are in part generated by the coordinated activity of millions of skin chromatophore organs, each of which is regulated by a set of chromatophore muscles directly innervated by centrally located chromatophore motoneurons. This study addresses the question of the identity and function of the transmitter(s) at the chromatophore neuromuscular junction (NMJ) in the European cuttlefish Sepia officinalis. Glutamate application causes a rapid contraction of the chromatophore muscles, resulting in chromatophore expansion. Pharmacological studies demonstrate that the chromatophore muscles contain receptors blocked by glutamate‐specific antagonists. Glutamate‐like immunoreactivity is also present in the somata of putative chromatophore motoneurons. These findings suggest that glutamate likely acts as a neurotransmitter at the chromatophore NMJ. Evidence is also presented suggesting that FMRFamide‐related peptides (FaRPs) also function as neurotransmitters at the Sepia chromatophore NMJ. FMRFamide application causes contraction of chromatophore muscles; however, the FMRFamide effect is slower and longer lasting than that of glutamate. Pharmacological data show that FMRFamide acts directly on the chromatophore muscles. FMRFamide‐immunopositive cells are present in the posterior chromatophore lobe, the putative location of the chromatophore motoneuron somata. A combination of immunocytochemistry and in situ hybridization shows that some putative chromatophore motoneurons express FaRP‐like immunoreactivity and an FaRP‐coding mRNA transcript. Many FMRFamide‐immunopositive cells in the posterior chromatophore lobes also express glutamate‐like immunoreactivity. We conclude that glutamate and FaRPs likely function as fast and slow transmitters, respectively, at the Sepia chromatophore NMJ. J. Comp. Neurol. 420:499–511, 2000.

Peripheral innervation patterns and central distribution of fin chromatophore motoneurons in the cuttlefish Sepia officinalis.

SUMMARY Body patterning behavior in unshelled cephalopod molluscs such as squid, octopuses, and cuttlefish is the ability of these animals to create complex patterns on their skin. This behavior is generated primarily by chromatophores, pigment-containing organs that are directly innervated by central motoneurons. The present study focuses on innervation patterns and location of chromatophore motoneurons in the European cuttlefish Sepia officinalis, specifically those motoneurons that control chromatophores of the fin. The fin is known to be innervated by the large, branching fin nerve. This study further characterizes the innervation of fin chromatophores by the fin nerve, generates a reference system for the location of fin nerve branches across individuals, and localizes the neurons whose axons innervate fin chromatophores through the fin nerve. Data from extracellular stimulation of fin nerve branches in intact animals demonstrate topographic innervation of fin chromatophores, while retrograde labeling data reveal the posterior subesophageal mass of the brain as the primary location of fin chromatophore motoneurons.

PEPTIDE DETECTION IN SINGLE CELLS USING A DOT IMMUNOBLOT ASSAY

A highly sensitive dot immunoblot assay (DIA) for the detection and quantitative measurement of small peptides in single cells is presented. This DIA protocol is simple, rapid, and produces no radioactive waste. Its femtomole sensitivity is 100 fold greater than previously described DIAs. This DIA method is sufficiently sensitive to allow reliable peptide measurements to be obtained from a single cell in a manner than is faster and easier than other peptide detection procedures. This method can also be used for several other purposes, including assessing antibody specificity and peptide quantification.