Isolation of neurosecretory granules containing cytochromogenic hormone activity from the corpora cardiaca of Blaberus discoidalis cockroaches

GENERAL

AND

COMPARATIVE

Isolation

ENDOCRINOLOGY

52, 149-156 (1983)

of Neurosecretory Granules Containing C~ochromoge~~ Hormone Activity from the Corpora Cardiaca of Blaberus discoidalis Cockroaches E. LAWSON-SMITH, TIMOTHY

Department

of Entomology,

AND L. L. KEELEY

K. HAY&,

Texas Agricultural College Station,

Experiment Station, Texas 77843

Texas A&M

University,

Accepted November 22, 1982 Neurosecretory granules (NSG) were isolated by differential centrifugation from a homogenate of the corpora cardiacs-corpora allata complex of newly emerged, adult, female Blaberus discoidalis cockroaches. By centrifuging at 11,OOOg,the procedure yielded a final pellet that was enriched in small, membrane-bound vesicles (103 nm mean diameter). This pellet contained 61% of the cytochromogenic activity present in the origin& homogenate at an 11-fold increase in concentration. A pellet formed at 40009 contained a mixture of mitochondria and larger NSG (146 nm mean diameter) and most of the hindgut-stimulating hormone activity. This procedure provided a quick, simple method for purifying neurosecretory granules and separating them into two categories of size and endocrine content.

Injections of corpora cardiaca (CC) homogenates stimulate the synthesis of cytohemes a + b during respiratory maturation of fat-body mitochondria in young adult Blaberus discoidalis cockroaches (Keeley, 1978). The active agent for stimulating cytoheme synthesis was designated as the cytochromogenic factor (CGF), and the properties determined for CGF indicate that it is a peptide (Hayes and Keeley, 1981). To determine if CGF was neuroendocrine in nature, a method was developed to isolate neurosecretory granules (NSG) with CGF activity. NSG are subcellular organelles that serve as storage vesicles for neurohormones in the axons of neurosecretory cells. In insects, NSG are abundant in the CC, which are neurohemal organs (Scharrer, 1963). The NSG are spherical, electron-dense, membrane-bound structures usually 100-300 nm in diameter (Maddrell, 1974).

Several centrifugation procedures have been employed to isolate NSG from insect neurosecretory tissures. Differential centrifugation procedures were applied to CC homogenates to produce NSG pellets with either cardioaccelerator activity (Evans, 1962) or adipokinetic activity (Stone and Mordue, 1979). Differential and ficoll-gradient centrifugation techniques were combined to isolate neurosecretory granules with hindgut-stimulating hormone ( activity (Sowa and Borg, 1975). Application of the previous procedures to the isolation of NSG from B. discoidalis proved unsatisfactory and, hence, a more efficient method is described in this paper, METHODS

AND MATERIALS

Experimental animals were cockroaches reared in wood shavings in 40-liter plastic buckets at 27” in a 12 hr light: 12 br dark circadian cycle with dog food and water provided ad lib. Animals were aged and segregated by sex from the day of emergence (day 0). Isolation of neurosecretory granules. Corpora cardiaca and corpora allata (CC + CA) were isolated from Experimental adult Blaberus

t To whom reprint requests and correspondence should be addressed.

animals. discoidalis

49 0016~6480/83 $1 SO Copyright All rights

@ 1983 by Academic Press, Inc. of reproduction in any form resewed

150

LAWSON-SMITH,

HAYES,

15 to 25, O-day-old, female insects and were pooled and homogenized in 0.4 ml of ice-cold 0.44 M sucrose in a glass homogenizer with a Teflon pestle. The glass homogenizer and transfer syringe were siliconized to reduce shear forces that might rupture NSG. The details of the isolation procedure are illustrated in Fig. 1. All pellets formed by centrifugation were resuspended in Ephrussi-Beadle Ringer (Ephrussi and Beadle, 1936). All preparations were frozen and thawed three times to rupture NSG and then centrifuged at 10,OOOgfor 3 min to remove tissue debris. The supernatants obtained by this last centrifugation were used in the assays for CGF, HSH, protein content, and radioactivity. Hindgut-stimulating hormone assay. The HSH assay was a modification of the procedure by Holman and Cook (1970). Hindguts from S-day-old B. discoidalis females were suspended in 14 ml of aerated insect saline solution at room temperature (Brown, 1965). The hindguts were exposed to an aliquot (0.1 CC pair equivalent) taken from each fraction formed by the NSG isolation procedure. The rate and amplitude of hindgut contractions were recorded and measured using an isotonic transducer (Holman and Cook, 1970) connected to a Houston Instrument recorder (10 mV). The hindgut activity of the original CC+CA homogenate

(15-25

CC-KX in 0.4 ml,

AND

KEELEY

was used as the basis for comparison with the activity in each pellet and in the final supematant. [35S]Cysteine incorporation into neurosecretory granzdes. Zero-day-old, female adult B. discoidalis were injected under the,posterior edge of the pronotal tergite in the middorsal line with 5 &i of [35S]cysteine (59 nmol) in 10 ~1 of Ephrussi-Beadle Ringer. The animals were allowed to incbrporate the [35S]cysteine for 3 hr at 27”. The radiolabeled CC + CA were extirpated and homogenized in 150 ~1 of ice-cold 0.44 M sucrose. A 50-~1 aliquot of the crude homogenate was removed for scintillation counting and for protein determination. The NSG were isolated from the remaining 100 ~1 of crude homogenate according to the procedure in Fig. 1. The resulting pellets were resuspended in 0.44 M sucrose. Aliquots were taken for liquid scintillation counting from the 600, 4000, and 11,OOOgpellets. AU samples were assayed for protein content by a microadaptation of the phenol method as reported by Layne (1956). The final supemantant was diluted I:1 with 10% trichloroacetic acid and centrifuged to separate the [35S]labeled proteins from unincorporated [35S]cysteine. The precipitated protein was then resuspended in the sucrose and assayed for protein content and radioactivity in the same manner as the other samples. AU samples for radioactivity assay

0.44

M sucrose)

(600 g, 10 min)

l---i

11,000

g pellet

::ELT 11,000 g supernatant (Final

Supernatant)

FIG. 1. Differential centrifugation procedure for isolating neurosecretory granules from homogenates of 15-25 corpora cardiacs-corpora allata complexes form O-day-old, adult Blaberus discoidalis.

CYTOCHROMOGENIC

HORMONE

were dissolved in a commercial tissue solubilizer at 45” for at least 1 hr and diluted in a commercial scintillation cocktail for counting. Electron microscopy studies. The structural compositions of the pellets formed during the isolation procedure were determined by electron microscopy. The pellets were prepared for fine structure studies as described by Stone and Mordue (1980). Assay for cytochromogenic activity. CGF activity in the fractions obtained from the NSG isolation procedure was assayed in decapitated animals by intrahemocoelic injections according to the 3-day regimen described previously (Hayes and Keeley, 1981). Cytocbrome synthesis was measured by the rate of [‘4tJaminolevulinic acid ([14CIALA) incorporation into cytohemes a and b. Cytohemes a and b were extracted from isolated fat-body mitochondria with acidic acetone according to the method of Basford et al. (1951).

FEW LTS Several of the published techniques for isolating NSG from insect neurosecretory structures were tried but were only marginally successful for our preparations. Initially, we tried the ultracentrifugation technique of Sowa and Borg (1975) but found poor recovery of HSH activity. We suspect that the long preparation times and high centrifugal forces needed for this procedure ruptured many of the NSG. We found that NSG prepared by, the high speed, differential centrifugation method of Stone and Mordue (1979) also gave poor recovery of HSH activity with most of the activity remaining in the post-NSG supernatant. However, our low recovery for HSH activity using this method agrees with their report that only 30% of the original adipokinetic hormone activity present in the CC was recovered in the final NSG pellet (Stone and Mordue, 1979). In an effort to improve recovery, we tried the lower speed, differential centrifugation procedure of Evans (1962). This method gave better yields of HSH activity, but ultrastructural examination of the NSG pellet showed extensive contamination by mitochondria and membranous debris. Visual examination of pellets formed by the Evans procedure showed an opalescent blue sub-

ACTIVITY

IN NSG

151

layer with a pale tan surface layer. The blue sublayer was reminiscent of the Tyndaljlll’s blue that result from the presence of NSG in the CC, whereas the tan surface Bayer resembled a mitochondrial pellet. Pellet Fine Structure We undertook to improve the purity of the NSG preparation formed by Evans’ method by adding a mitochondrial is&&n step at 4000g for 15 min (Fig. I) before the NSG isolation step. Ultrastructural examination of pellets formed by our method showed that the NSG pellet was free of most of the cellular debris that we observed in Evans’ pellet (Fig. 2b). Further structural analysis of the 4000g pellet confirmed the abundance of mitochondria (Fig. 2a). NSG were also frequent in the 4000g pelle ong with mitochondria and a structure LlElknown origin or function. This latter strtfcture resembled a group within a single membrane. these structures may be the same manner as microsomes by disrupted membranes fusing with themselves and enclosing NSG in the process. Ultrastructural examination of the 6OOg pellet (not shown) revealed nuclei and cellular debris. Measurements of the NSG profiles in the 4000 and 11,OOOg pellets the NSG present. in the two p significantly in size. NSG present in the 4@Xlg pellet ranged in size from 73 to 209 nm with a mean 2 SE of 146 +- 6 nm for 29 individual NSG profiles counted. In contrast, the NSG present in the 11,OOOgpellet ranged form 70 to 157 nm with a mean & SE of 103 i 4 nm, also for 29 replicate NSG profiles. Therefore, the isolation method described here can resolve the NSG into two size categories. Distribution Hormone

~~pjFindgut-Stimulating Activity

HSH is a neurohormone found in the CC (Holman and Marks, 1974). Sowa and Borg

1.52

LAWSON-SMITH,

HAYES,

AND KEELEY

FIG. 2. Fine structural composition of the pellets formed by differential centrifugation of a homogenate of 15-20 corpora cardiacs-corpora allata from O-day-old adult, female Blaberus discoidalis. (a) 4000g pellet, arrow denotes unknown structure containing a multiple NSG; (b) 1l,OOOg pellet.

(1975) used HSH activity to identify their NSG fraction. We also used this bioassay since it is direct and easy to perform. Our NSG isolation technique produced unexpetted results with the HSH assay. Most of

the HSH activity was found in the 4000g pellet (Fig. 3) with little HSH activity in the putative NSG pellet formed at 11,OOOg. The supernatant also contained high HSH activity which indicated a considerable de-

CYTOCHROMOGENIC

HORMONE

FIG. 3. Contractile activity of in vitro hindguts from Sday-old, adult female Blaberus discoidalis in response to hindgut-stimulating hormone activity in preparations of corpora cardiaca and corpora allata. (a) Untreated; (b) Treatment with 0.1 gland-pair equivalent of crude gland homogenate; (c) Treatment with 0.1 gland-pair equivalent from 600g pellet; (d) Treatment with 0.1 gland-pair equivalent from 4000g pellet; (e) Treatment with 0.1 gland-pair equivalent from 11,OOOgpellet; (f) Treatment with 0.1 gland-pair equivalent from final supernatant; (g) Treatment with 0.4 gland-pair equivalent from 11,OOOgpellet. The verticle calibration bar equals a 2-mm hindgut contraction. The horizontal calibration bar equals 2 min.

gree ofdisruption of HSH-containing NSG. The HSH assay is difficult to quantitate, but it appears from the data (Fig. 3) that the 4QOOg pellet contained roughly six to seven times more HSH activity than did the 11 ,OOOg pellet. [35S]Cysteine Zncorporation. Some neurosecretory materials contain a high proportion of cysteine (Schreiner, 1966; Mordue and Goldsworthy, 1969; Durnberger et al, 1978; Friedel et al, 1980). The content of cysteine sulfhydryl groups is reported to be the basis for the staining methods that identify neurosecretory cells (Sloper, 1957;

ACTIVITY

IN NSG

153

Schreiner, 1966; Prento, 1972; Rowell, 1976), and [35S]cysteine labeling of neurosecretory materials is used to study t synthesis-secretion cycles for neurohormones in several insect species (Highnam and Mordue, 1970; Schooneveld, 1970; Highnam, 1971; Mordue and Hi 1973; Highnam and Mordue, 1974). Th concentration of cysteine ~found in th is believed to be mainly a function of proteins associated with the ~eurobormo~~s rather than of the hormones per se. Incorporation of C3YS]cysteine is rep into specific neurosecretory into general cellular proteins (Friedel and Loughton, 1980). The purported specificity of ]“5S]cysteine for labeling neurosecretory materials suggested that specific labeling may be a useful marker for identifying the NSG-containing fractions formed by our isolation procedure. Preliminary studies were ~e~~rrned to show the label incorporation pattern intact CC of 3. discoidalis. These stu showed that 59 nmol of [35S]cysteine (6.5 t&i) incorporated into TCA-~rec~p~tab~~ proteins of the glands at a linear rate for u to 4 hr, and O-day-old animals had the highest rate of incorporation. For t reason, 3 hr was chosen for labeling of NSG to ensure maximal label i~co~o~ati~~ within the linear period. The data show that no fraction from our NSG isolation procedure contained a significantly high content of [35S]cysteine relative to other fractions (Table I). Therefore, [35S]cysteine lacelike is not sufficiently specific that it can as a means for identifying fraction, taining a high proportion of NSG. Distribution of CGF Activity CGF activity was correlated to the fractions formed by our isolation proce Most CGF activity was localized in the pellet formed at 11 ,OOOg and showed an 1 Ifold concentration with a 61% recovery of the original homogemate activity (Table I). The 4OOOg pellet was the only other fraction

LAWSON-SMITH,

154

HAYES, TABLE

DISTRIBUTION

AND KEELEY

1

OF CGF AND 35S-L~~~~~~ PEETIDES IN CELL FRACTIONS ISOLATED FROM 15 CORPORA CORPORA ALLATA COMPLEXES OF O-DAY-OLD, ADULT FEMALE Blaberus discoidalis

Units of CGF activity0 * range

Fraction Crude homogenate 600g Pellet 4000g Pellet 11,OOOgPellet 11,OOOgSupernatant

104 k 6-t-3 25 1: 63 ” 12 ”

5 3 5 3

Protein bJ&

Units of activity per mg protein

Total CGF activity m

357 171 62 19 120

291 35 403 3316 100

100 6 24 61 12

CARLNACA-

35S-Labelingb (dpm/mg protein) + SE 20,066 11,789 14,033 13,667 6,666

i 8,824 I 6,807 i 5,099 -t 5,487 -t- 333

a One CGF unit = 0.08 corpora cardiaca, the minimum dose that causes a maximum cytochromogenic response. CGF values are based on the mean for two replicate NSG isolations. Each CGF assay typically consisted of eight replicate animals. b Values represent an average of three replicate NSG isolations.

of the preparation that contained any significant CGF activity. Therefore, CGF activity is associated predominantly with the smaller NSG. DISCUSSION

The differential centrifugation procedure described here is a simple, quick procedure for isolating NSG from the insect neurosecretory system. The method provides a preparation of purified particles in the 11,OOOgpellet that meet the criteria for NSG based on their size range and structural appearance (Sharrer, 1963; Sowa and Borg 1975; Stone and Mordue 1979). Although the particles in this pellet fall within the range for NSG (70-300 nm), their sizes tend toward the lower end of the range. An unexpected result was that CGF activity was associated, especially, with this preparation of smaller granules, whereas HSH activity was found principally in the 4000g fraction which contained larger NSG. The lack of specific labeling by [35S]cysteine is contradictory to much of the literature on neurosecretion. Our preliminary finding that newly emerged adult females incorporated [35S]cysteine into the CC proteins more actively than did other ages suggested that neurosecretory materials were being synthesized at this time. However, near uniformity for the specific activities in

all of the isolated fractions suggests the [35S]cysteine did not specifically label neurosecretory materials but was incorporated, generally, into cellular proteins of the CC. Histological observations on the CC of male B. discoidalis suggested a 35% enlargement of the glands during the first 24 hr of adult life based on a decrease in the number of nuclei/millimeters squared (Keeley, unpublished observation). This suggested cellular hypertrophy occurs in the CC during the first day of adult life. Thereafter, the nuclei/unit area remained essentially constant, at least through 10 days of adult age. Based on this apparent period of early cellular growth, [35S]cysteine incorporation into the CC on day 0 may have resulted form the cellular growth, not the synthesis of neurosecretory materials. Therefore, general cellular proteins were labeled to such an extent that they were not distinguishable from neurosecretory proteins when based on specific activity. It is also likely that the NSG present in the glands on day 0 were already there at emergence and not synthesized de novo, especially during the brief 3-hr period of label incorporation used in these studies. We might have found label incorporation more specifically into NSG, as reported in the literature, had we used older test insects. Our isolation technique appeared to ef-

CYTOCHROMOGENIC

HORMONE

fectively separate two sizes of particles, both of which resembled published examples of NSG and both of which fell into the accepted size ranges for NSG. Therefore, we concluded at this point that the pellets formed at both 4000 and 1 I ,OOOg contained NSG. The larger NSG in the 4000g pellet coisolated with the mitochondria and appeared to contain the major amount of HSH activity. In contrast, the 11 ,OOOg pellet was less contaminated by cellular debris and consisted of small NSG that were high in CGF activity. Both the 4000 and 11 ,OOOg pellets contained some HSH and CGF activities indicating cross-contamination. Cross-contamination of fractions is expected in any differential centrifugation procedure. Approximately 30% of the CGF activity was lost into the 4000g pellet, but the nearly 60% of the CGF activity present in the 11 ,OOOg pellet was associated with relatively clean NSG. Very little CGF activity was found in the final supernatant which indicat.es very little rupture of the CGF-containing NSG. In conclusion, our modified isolation procedure provides a simple, reasonably quick method for isolating NSG, and it has resulted in several insights into the nature of CGF. Specific [YS]cysteine labeling of the fraction with CGF activity did not occur, and it appears that not all NSG contain a significant cysteine content since it is known that not all NSG are PAF-positive. The structural appearance and size range of the granules in the I l,OOOg pellet suggest that the granules are of neuroendocrine origin. Although the NSG isolated with CGF activity are in the smaller range of NSG and within the range of biogenic amine-containlng granules (30-200 nm diameter (Evans, 1980), reversed-phase TLC indicates that CGF does not migrate with biogenie amines (unpublished observations). Hence, CGF activity appears associated with “small” NSG. The association of CGF with a NSG fraction confirms the endocrine nature of CGF and demonstrates that it is

ACTIVITY

IN NSG

155

not a general tissue component. Therefore, due to its endocrine origin, we propose that, henceforth, the “cytochromogenic factor” be referred to as the cytochromogenic hsrmone . ACKNOWLEDGMENTS The authors thank Mr. Dave Lee W~~iarns for his competent technical assistance and Dr. B. J. Cook (Veterinary Toxicology and Entomology Kesearch Laboratory, USDA, College Station) for the loan of the isotonic transducer for measuring hindgut comractions. The research presented in this report was supported by NIH Grant TMP AI 15190, NSF Grant PCM 81-03277 to L.L.K, and by the Texas Agricultural Experiment Station.

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