Ncuroendocrinology 19: 12-27 (1975)
An Ultrastructural and Radioimmunoassay Study of Anterior Pituitary Somatotrophs Following Pituitary Portal Vessel Infusion of Growth Hormone Releasing Factor1 D. L. W il b u r 2, W.C. W o r t h in g t o n , jr. and R.R. M a r k w a l d 2 Department of Anatomy. Medical University of South Carolina, Charleston, S.C.
Key Words. Growth hormone • Growth hormone-releasing factor • Hypophysial infusion Abstract. The hypophysial portal vessels and anterior pituitary glands of adult male rats were surgically exposed, cannulated and infused for 1 min with saline, growth hor mone-releasing factor (GH-RF), and dbcAMP. After cessation of infusion, anterior pi tuitary glands were collected at 1,5, 15, 30 or 60 min for electron microscopic and ultrastructural cytochemical examination. Before and after cannulation of a portal vessel a 1-ml sample of blood was collected at 1,5, 15, 30 or 60 min from the femoral vein for RIA of growth hormone. When viewed ultrastructurally, the initial response following the infusion of GH-RF into a portal vessel was one of granule release. Emiocytic activity was observed at all time intervals studied. This response was followed 30 min later by evidence of in creased protein synthesis. Significant increases in plasma GH levels were present at 1,5, and 15 min following infusion of GH-RF but not at 30 or 60 min. Preliminary analysis of the RIA data suggests that dbcAMP was significantly more potent than GH-RF in elevating radioimmunoassayable plasma GH levels. The results suggest that similar mechanisms of synthesis and release were involved.
In our previous ultrastructural studies on the mechanism(s) by which dibutyryl adenosine-3'-5'-cyclic phosphate monosodium (dbcAMP) stim ulates growth hormone (GH) release and synthesis [W ilbu r et a!., 1974], we found the response of somatotrophs to be extremely rapid and a maximal emiocytic effect was observed 1 min after infusion. This initial effect of dbcAMP was followed 15-30 min later by ultrastructural evidence of in-
Received: March 7th, 1975; revised MS accepted: June 24th, 1975.
Downloaded by: Boston University 128.197.229.194 - 1/10/2019 11:50:03 PM
1 Supported by NIH research grant 5 R01 AM 10520. 2 Present address: Department of Anatomy, School of Medicine, Texas Tech Uni versity, Lubbock, TX 79409.
W ilbur/W orthington/M arkwald
13
creased protein synthesis. These results, coupled with the findings of similar morphologic alterations in somatotrophs following the systemic injection of hypothalamic extracts and growth hormone-releasing factor (GH-RF) [C oates et al., 1971a, b], suggested that GH-RF and dbcAMP may work by similar mechanism(s) of action on somatotrophs. To test this hypothesis in our in vivo test system, GH-RF obtained from two independent sources was infused into hypophysial portal vessels; its effects were viewed ultrastructurally and measured by radioimmunoassay.
Adult male Wistar rats weighing 200-240 g were housed in an environmentally con trolled room with 2 rats to a cage, each maintained by Purina laboratory food and water ad libitum. The animals were anesthetized with 10% urethane (1,200 mg/kg). The anterior pituitary gland and hypophysial portal vessels were surgically exposed according to the method described by W orthington [1955]. A pneumatic DeFonbrune micro-manipulator was used to place the microcannula in a portal vessel. A Palmer injection pump regulated the flow of the infusate through the microcannula at a rate of 1-2 /tl/min [Porter et al., 1970]. The medium for the secretagogues was physiological saline buffered to pH 7.4. A small amount of lissamine green was added to aid in determining the distribution of the infused portal vessel. A synthetic growth hormone-releasing hormone (GH-RH) (H-Val-His-Leu-Ser-AIa-Glu-Glu-Lys-GluAla-OH) prepared by Dr. R.M.S. N air and characterized by Schally et al. [1971] was dissolved in the medium to give a final concentration of 1.4 x I0~ 7 m and infused for I min. A synthetic GH-RF, purchased from Spectrum Medical Industries, Inc. (product no. 250850) and having the same amino acid sequence, was dissolved in the medium to yield a final concentration of 2.8 x 10~7 m and was infused for I min. N 6-02'-Dibutyryl adenosine-3'-5'-cyclic phosphate monosodium-5-H20 (dbcAMP, MW 582.6, Calbiochcm) was dissolved in the medium to give a final concentration of 4.6 x 10“ 7 m dbcAMP. Tissue was collected after sacrificing animals by guillotine at 1,5, 15, 30 or 60 min after infusion. There were 4 experimental animals in each time period. Controls consisted of sham-operated animals without infusion, unoperated animals, animals infused with saline and lissamine green only, and tissue from infused glands in an area outside the distribution of the infused vessel. Anterior pituitary glands were separated into infused portions and noninfused portions and then cut into 1-mm-1 pieces. Tissue was fixed in 6% glutaraldehyde with 1% hydrogen peroxide in 0.1 m cacodylate butler, pH 7.3, for 1 h at room temperature and then immersed in 6% glutaraldehyde in 0.1 m cacodylate buffer without H20 2 and fixed for 1 h at 4 JC. The tissue was subsequently rinsed, postfixed in cacodylate-buffered 1% O s04, dehydrated in graded alcohols, embedded in Epon 812, sectioned, doubly stained with uranyl acetate and lead citrate [Venable and C oggeshall, 1965], and viewed with a Hitachi HU-1 IE microscope. Semi-thin sections, stained with toluidine blue, were used to identify cor responding thin sections.
Downloaded by: Boston University 128.197.229.194 - 1/10/2019 11:50:03 PM
Materials and Methods
14
W ilbur/W orthington/M arkwald
For identifying lysosomes, the presence of reaction product for acid phosphatase was used as a marker. Anterior pituitary tissue was fixed for 1 h in 2% redistilled and purified glutaraldehyde in 0.1 m cacodylate buffer, rinsed 16 h in cold buffer, incubated 30 min at 37°C in a modified G omori [1952] medium for acid phosphatase (7.8 him P b N 0 3 and 22.4 mM sodium fi glycerophosphate in 0.1 m tris maleate, pH 5.0), rinsed, postfixed in OsO., and embedded in Epon 812 as described above. Control procedures included omis sion of substrate, substitution of substrate with ATP, or addition of 10~2 m NaF to the complete medium. For identifying the Golgi apparatus, the presence of reaction product for thiamine pyrophosphatase was used as a marker enzyme. Anterior pituitary tissue was fixed for 2 h in 2.7% glutaraldehyde in 0.1 m cacodylate buffer at 4°C and rinsed overnight in 0.1 m cacodylate buffer plus 7% sucrose. It was then incubated for 30 min at 37°C in a modified N ovikoff and G oldfischer [1961] medium for TPPase (3.6 him PbNOj, 7.5 mg thiamine pyrophosphate, 0.025 him manganese chloride in 0.2 m tris maleate buffer, pH 7.0), rinsed, postfixed in OsO* and embedded in Epon 812 as described above. Control procedures included omission of substrate, substitution of substrate with ADP, or addition of 10-2 m NaF to the complete medium. Table l. Summary of GH-RF infusion into portal vessels for I min (1.4 x 10~7 moles/min) Rough Golgi Endoplasmic Reticulum
Control
Lamellar and Polar Lamellar and Polar Lamellar and Polar
GH-RF 1
GH-RF 5
GH-RF 15 Lamellar and Diffuse GH-RF 30 Greatly Distended and Diffuse GH-RF 60
Distended and Diffuse
Number of Secretory Granules
Number Granule of DistribuLysosomal tion Bodies
Prominent Undilated
—
—
Prominent Undilated
—
Prominent Many Vesicles — Microtubules Frequently Hypertrophied Many Vesicles Microtubules Marked Hypertrophy Many Vesicles Microtubules Prominent Undilated
Some 1ncreased Some Decreased Some Increased Some Decreased —
—
—
4-
Amount of Emiocytosis
Scattered — Some Peripheral Massive Scattered Many Some Peripheral Multiple Massive Scattered Many More Peripheral Multiple + ++ Scattered More Peripheral More Peripheral Scattered Many Peripheral
4- 4*
4-
4Downloaded by: Boston University 128.197.229.194 - 1/10/2019 11:50:03 PM
Tissue
Hypophysial Portal Infusion of GH-RF
15
Serum GH was determined by double antibody precipitation, modified from the methods of Parker et at. [1965]. Rat GH standards were prepared from rat GH standard lot R965B obtained from Dr. A. E. W ilhelmi. The first antibody used in these studies was NIH-monkey-anti-rat GH. The second antibody used in these studies was goat-anti monkey y globulin purchased from Antibodies Inc., Davis, Calif. All serum values were determined as ng of the R965B standard per ml.
Results (I) Electron Microscopic Studies ( table I)
Fig. 1. Somatotrophs from sham-operated control animal. Rough endoplasmic reticu lum (RER), Golgi complexes (G), secretory granule(s) size and distribution are charac teristic of somatotrophs from control animals. GlutaraIdehyde-H20 2 fixation: stained with lead citrate and UA. * 11,280.
Downloaded by: Boston University 128.197.229.194 - 1/10/2019 11:50:03 PM
(A) Control Animals A typical ovoid somatotroph, observed in all groups of control animals, is shown in figure 1, and corresponds with other descriptions [R in eh a r t and
16
W ilbur/W orthington /M arkwald
F a r q u h a r , 1953; L ever and P eterson , I960; F a r q u h a r , 1961; C ostoff .
Fig. 2. Somatotroph from unoperated control animal. Golgi lamellae are extensive but non-dilated and displayed little thiamine pyrophosphatase activity. Glutaraldehyde-H20 2 fixation; stained with UA only, x 33,600.
Downloaded by: Boston University 128.197.229.194 - 1/10/2019 11:50:03 PM
1973], Rough endoplasmic reticulum (RER) was extensive, generally nondistended and polarized to one surface. An exhaustive examination of nu merous sections revealed that only vacuoles and vesicles were associated with the Golgi complex. The Golgi lamellae were extensive but nondilated and displayed little thiamine pyrophosphatase activity. Secretory granules ap peared numerous and were distributed heterogeneously. Granules varied in diameter from 200-400 m/f. Granule extrusion was rarely encountered in glands from untreated and sham-operated animals. Controls infused with saline exhibited more emiocytic activity, but the number of emiocytic vesicles was small and varied from cell to cell. Acid phosphatase activity was restricted primarily to condensing vacuoles and primary lysosomes. Thiamine pyro phosphatase activity was sparse and limited to the inner Golgi cisternae (fig. 2). Condensing vacuoles were usually free of reaction deposits.
Hypophysial Portal Infusion of GH-RF
17
(B ) Animals Infused with GH-RF Two separate and independent sources of a synthetic decapeptide with known growth hormone releasing activity were used (Nair GH-RF and Spectrum GH-RF). Because the morphological changes occurring after the infusion of either one of the GH-RFs were indistinguishable, the results will be presented collectively. All animals were infused for I min.
Fig. 3. Somatotrophs 1 min following 1-min infusion of GH-RF displayed emiocytic activity often with 2 or more granules coalescing and emerging through a common channel (long arrows). Empty vesicles are also seen at the cell membrane (short arrow). Glutaraldehyde fixation; stained with lead citrate and UA. x 20,000.
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(!) 1-min Postinfusion Group Areas of multiple granule extrusion occurred I min following infusion of GH-RF (fig. 3). The RER was lamellar and polar as seen in controls. Granules remained dispersed throughout the cell with no specific alignment near the plasma membrane. Granules, when extruded into the perivascular space or basement membrane area, usually tended to be less dense, had less sharply defined margins, and appeared to show less of the limiting membrane. Acid phosphatase and TPPase activity was similar to infused controls.
18
W ilbur/W orthington/M arkwald
(2) 5-mirt Postinfusion Group The RER remained similar to that observed in controls. Many small smooth-surfaced and coated vesicles appeared in the proximity of the Golgi complex in close association with microtubules (fig. 4). Emiocytic activity continued to be extensive, showing many sites of multiple release. Occasional images were recorded of multiple granule cores appearing simultaneously within a plasmalemmal invagination opening into intercellular or pericapillary spaces. Granule number and distribution were similar to those observed in controls.
Fig. 4. Somatotrophs 5 min after 1-min infusion of GH-RF. Many small smooth surfaced and coated vesicles appeared near the Golgi complex in close association with microtubules (arrows). Glutaraldehyde-H,(); fixation; stained with lead citrate and UA. x 18,240.
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(3) 15-min Postinfusion Group Golgi complexes appeared more hypertrophied than those from the pre vious groups. Microtubules near smooth-surfaced and coated vesicles were
Hypophysial Portal Infusion ofG H -RF
19
also seen near the Golgi complex (fig. 5). The RER was unchanged from controls and remained lamellar and diffuse. Although the granule content of individual somatotrophs varied, overall levels of granulation resembled those seen in controls. Coated invaginations were occasionally observed at plasma membranes. Emiocytic activity was present, although not as massive as in the 2 earlier time groups. Acid phosphatase activity was similar to control levels, as was TPPase activity.
Fig.5. Somatotroph 15 min following 1-min infusion of GH-RF. Numerous micro tubules (long arrows) and newly synthesized secretory granules (short arrows) are seen near the Golgi complex. Glutaraldehyde-H20 2 fixation; stained with lead citrate and UA. x 24,000.
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(4) 30-min Postinfusion Group Golgi complexes were vesicular and displayed elevated TPPase activity (fig. 6). Secretory granules continued to be observed within the Golgi com plex. The RER was distended and diffuse throughout the cell (fig. 7). Extreme variation in secretory granule number and distribution was evident on com-
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20 W ilbur/W orthington /M arkwald
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Hypophysial Portal Infusion of GH-RF 21
22
W ilbur/W orthington /M arkwald
parison with earlier time groups. Emiocytic activity was less than that ob served at 1,5, and 15 min postinfusion. An increase in acid phosphatase ac tivity was observed in this time group. (5) 60-min Postinfusion Group Golgi complexes were similar to those observed in the previous 30-min group (fig. 8). The RER was not as distended as it was in the 30-min group, but it was more distended and diffuse than observed in controls. The number of secretory granules and their distribution were similar to observations of earlier time groups. Emiocytic activity was still present. An increase in the number of multivesicular and multilamellar bodies, along with acid phos phatase reactive lysosomes (fig. 9), was present in this group. TPPase activity was not elevated above control levels.
See Fig. 6-9, pages 20 and 21 Fig. 6. Somatotrophs 30 min following 1-min infusion of GH-RF continued to de monstrate distended and diffuse RER throughout the cell. Glutaraldehyde-H20 2 fixation; stained with lead citrate and UA. x 24,000. Fig. 7. Thiamine pyrophosphatase activity in somatotroph 30 min following 1-min infusion of GH-RF. Glutaraldehyde fixation; stained with UA only, x 38,000. Fig. 8. Somatotrophs 60 min following 1-min infusion of GH-RF. The Golgi complexes were vesicular and hypertrophied in somatotrophs containing few numbers of secretory granules. At this same time period in somatotrophs containing increased secretory granules, the Golgi complex resembled controls'. Glutaraldehyde-H20 2 fixation; stained with lead citrate and UA. x 24,000. Fig. 9. Acid phosphatase activity in a somatotroph 60 min following 1-min infusion of GH-RF. Gomori method. An increase in the number of acid phosphatase reactive granules (arrows) was observed at this time. Glutaraldehyde fixation; stained with UA only, x 48,000.
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(II) Radioimmunoassay o f Rat Growth Hormone (table II) A two-way analysis of variance followed by the calculation of the appro priate least significant differences [LSD (0.5) Appendix II] was used to deter mine if significant differences were present among the various treatment methods (dbcAMP vs GH-RFs: NGH-RF vs SGH-RF) and between preand postinfusion samples. (1) Following the 30-min infusion of dbcAMP, postinfusion levels of radioimmunoassayable plasma GH were significantly elevated (p
An Ultrastructural and Radioimmunoassay Study of Anterior Pituitary Somatotrophs Following Pituitary Portal Vessel Infusion of Growth Hormone Releasing Factor1 D. L. W il b u r 2, W.C. W o r t h in g t o n , jr. and R.R. M a r k w a l d 2 Department of Anatomy. Medical University of South Carolina, Charleston, S.C.
Key Words. Growth hormone • Growth hormone-releasing factor • Hypophysial infusion Abstract. The hypophysial portal vessels and anterior pituitary glands of adult male rats were surgically exposed, cannulated and infused for 1 min with saline, growth hor mone-releasing factor (GH-RF), and dbcAMP. After cessation of infusion, anterior pi tuitary glands were collected at 1,5, 15, 30 or 60 min for electron microscopic and ultrastructural cytochemical examination. Before and after cannulation of a portal vessel a 1-ml sample of blood was collected at 1,5, 15, 30 or 60 min from the femoral vein for RIA of growth hormone. When viewed ultrastructurally, the initial response following the infusion of GH-RF into a portal vessel was one of granule release. Emiocytic activity was observed at all time intervals studied. This response was followed 30 min later by evidence of in creased protein synthesis. Significant increases in plasma GH levels were present at 1,5, and 15 min following infusion of GH-RF but not at 30 or 60 min. Preliminary analysis of the RIA data suggests that dbcAMP was significantly more potent than GH-RF in elevating radioimmunoassayable plasma GH levels. The results suggest that similar mechanisms of synthesis and release were involved.
In our previous ultrastructural studies on the mechanism(s) by which dibutyryl adenosine-3'-5'-cyclic phosphate monosodium (dbcAMP) stim ulates growth hormone (GH) release and synthesis [W ilbu r et a!., 1974], we found the response of somatotrophs to be extremely rapid and a maximal emiocytic effect was observed 1 min after infusion. This initial effect of dbcAMP was followed 15-30 min later by ultrastructural evidence of in-
Received: March 7th, 1975; revised MS accepted: June 24th, 1975.
Downloaded by: Boston University 128.197.229.194 - 1/10/2019 11:50:03 PM
1 Supported by NIH research grant 5 R01 AM 10520. 2 Present address: Department of Anatomy, School of Medicine, Texas Tech Uni versity, Lubbock, TX 79409.
W ilbur/W orthington/M arkwald
13
creased protein synthesis. These results, coupled with the findings of similar morphologic alterations in somatotrophs following the systemic injection of hypothalamic extracts and growth hormone-releasing factor (GH-RF) [C oates et al., 1971a, b], suggested that GH-RF and dbcAMP may work by similar mechanism(s) of action on somatotrophs. To test this hypothesis in our in vivo test system, GH-RF obtained from two independent sources was infused into hypophysial portal vessels; its effects were viewed ultrastructurally and measured by radioimmunoassay.
Adult male Wistar rats weighing 200-240 g were housed in an environmentally con trolled room with 2 rats to a cage, each maintained by Purina laboratory food and water ad libitum. The animals were anesthetized with 10% urethane (1,200 mg/kg). The anterior pituitary gland and hypophysial portal vessels were surgically exposed according to the method described by W orthington [1955]. A pneumatic DeFonbrune micro-manipulator was used to place the microcannula in a portal vessel. A Palmer injection pump regulated the flow of the infusate through the microcannula at a rate of 1-2 /tl/min [Porter et al., 1970]. The medium for the secretagogues was physiological saline buffered to pH 7.4. A small amount of lissamine green was added to aid in determining the distribution of the infused portal vessel. A synthetic growth hormone-releasing hormone (GH-RH) (H-Val-His-Leu-Ser-AIa-Glu-Glu-Lys-GluAla-OH) prepared by Dr. R.M.S. N air and characterized by Schally et al. [1971] was dissolved in the medium to give a final concentration of 1.4 x I0~ 7 m and infused for I min. A synthetic GH-RF, purchased from Spectrum Medical Industries, Inc. (product no. 250850) and having the same amino acid sequence, was dissolved in the medium to yield a final concentration of 2.8 x 10~7 m and was infused for I min. N 6-02'-Dibutyryl adenosine-3'-5'-cyclic phosphate monosodium-5-H20 (dbcAMP, MW 582.6, Calbiochcm) was dissolved in the medium to give a final concentration of 4.6 x 10“ 7 m dbcAMP. Tissue was collected after sacrificing animals by guillotine at 1,5, 15, 30 or 60 min after infusion. There were 4 experimental animals in each time period. Controls consisted of sham-operated animals without infusion, unoperated animals, animals infused with saline and lissamine green only, and tissue from infused glands in an area outside the distribution of the infused vessel. Anterior pituitary glands were separated into infused portions and noninfused portions and then cut into 1-mm-1 pieces. Tissue was fixed in 6% glutaraldehyde with 1% hydrogen peroxide in 0.1 m cacodylate butler, pH 7.3, for 1 h at room temperature and then immersed in 6% glutaraldehyde in 0.1 m cacodylate buffer without H20 2 and fixed for 1 h at 4 JC. The tissue was subsequently rinsed, postfixed in cacodylate-buffered 1% O s04, dehydrated in graded alcohols, embedded in Epon 812, sectioned, doubly stained with uranyl acetate and lead citrate [Venable and C oggeshall, 1965], and viewed with a Hitachi HU-1 IE microscope. Semi-thin sections, stained with toluidine blue, were used to identify cor responding thin sections.
Downloaded by: Boston University 128.197.229.194 - 1/10/2019 11:50:03 PM
Materials and Methods
14
W ilbur/W orthington/M arkwald
For identifying lysosomes, the presence of reaction product for acid phosphatase was used as a marker. Anterior pituitary tissue was fixed for 1 h in 2% redistilled and purified glutaraldehyde in 0.1 m cacodylate buffer, rinsed 16 h in cold buffer, incubated 30 min at 37°C in a modified G omori [1952] medium for acid phosphatase (7.8 him P b N 0 3 and 22.4 mM sodium fi glycerophosphate in 0.1 m tris maleate, pH 5.0), rinsed, postfixed in OsO., and embedded in Epon 812 as described above. Control procedures included omis sion of substrate, substitution of substrate with ATP, or addition of 10~2 m NaF to the complete medium. For identifying the Golgi apparatus, the presence of reaction product for thiamine pyrophosphatase was used as a marker enzyme. Anterior pituitary tissue was fixed for 2 h in 2.7% glutaraldehyde in 0.1 m cacodylate buffer at 4°C and rinsed overnight in 0.1 m cacodylate buffer plus 7% sucrose. It was then incubated for 30 min at 37°C in a modified N ovikoff and G oldfischer [1961] medium for TPPase (3.6 him PbNOj, 7.5 mg thiamine pyrophosphate, 0.025 him manganese chloride in 0.2 m tris maleate buffer, pH 7.0), rinsed, postfixed in OsO* and embedded in Epon 812 as described above. Control procedures included omission of substrate, substitution of substrate with ADP, or addition of 10-2 m NaF to the complete medium. Table l. Summary of GH-RF infusion into portal vessels for I min (1.4 x 10~7 moles/min) Rough Golgi Endoplasmic Reticulum
Control
Lamellar and Polar Lamellar and Polar Lamellar and Polar
GH-RF 1
GH-RF 5
GH-RF 15 Lamellar and Diffuse GH-RF 30 Greatly Distended and Diffuse GH-RF 60
Distended and Diffuse
Number of Secretory Granules
Number Granule of DistribuLysosomal tion Bodies
Prominent Undilated
—
—
Prominent Undilated
—
Prominent Many Vesicles — Microtubules Frequently Hypertrophied Many Vesicles Microtubules Marked Hypertrophy Many Vesicles Microtubules Prominent Undilated
Some 1ncreased Some Decreased Some Increased Some Decreased —
—
—
4-
Amount of Emiocytosis
Scattered — Some Peripheral Massive Scattered Many Some Peripheral Multiple Massive Scattered Many More Peripheral Multiple + ++ Scattered More Peripheral More Peripheral Scattered Many Peripheral
4- 4*
4-
4Downloaded by: Boston University 128.197.229.194 - 1/10/2019 11:50:03 PM
Tissue
Hypophysial Portal Infusion of GH-RF
15
Serum GH was determined by double antibody precipitation, modified from the methods of Parker et at. [1965]. Rat GH standards were prepared from rat GH standard lot R965B obtained from Dr. A. E. W ilhelmi. The first antibody used in these studies was NIH-monkey-anti-rat GH. The second antibody used in these studies was goat-anti monkey y globulin purchased from Antibodies Inc., Davis, Calif. All serum values were determined as ng of the R965B standard per ml.
Results (I) Electron Microscopic Studies ( table I)
Fig. 1. Somatotrophs from sham-operated control animal. Rough endoplasmic reticu lum (RER), Golgi complexes (G), secretory granule(s) size and distribution are charac teristic of somatotrophs from control animals. GlutaraIdehyde-H20 2 fixation: stained with lead citrate and UA. * 11,280.
Downloaded by: Boston University 128.197.229.194 - 1/10/2019 11:50:03 PM
(A) Control Animals A typical ovoid somatotroph, observed in all groups of control animals, is shown in figure 1, and corresponds with other descriptions [R in eh a r t and
16
W ilbur/W orthington /M arkwald
F a r q u h a r , 1953; L ever and P eterson , I960; F a r q u h a r , 1961; C ostoff .
Fig. 2. Somatotroph from unoperated control animal. Golgi lamellae are extensive but non-dilated and displayed little thiamine pyrophosphatase activity. Glutaraldehyde-H20 2 fixation; stained with UA only, x 33,600.
Downloaded by: Boston University 128.197.229.194 - 1/10/2019 11:50:03 PM
1973], Rough endoplasmic reticulum (RER) was extensive, generally nondistended and polarized to one surface. An exhaustive examination of nu merous sections revealed that only vacuoles and vesicles were associated with the Golgi complex. The Golgi lamellae were extensive but nondilated and displayed little thiamine pyrophosphatase activity. Secretory granules ap peared numerous and were distributed heterogeneously. Granules varied in diameter from 200-400 m/f. Granule extrusion was rarely encountered in glands from untreated and sham-operated animals. Controls infused with saline exhibited more emiocytic activity, but the number of emiocytic vesicles was small and varied from cell to cell. Acid phosphatase activity was restricted primarily to condensing vacuoles and primary lysosomes. Thiamine pyro phosphatase activity was sparse and limited to the inner Golgi cisternae (fig. 2). Condensing vacuoles were usually free of reaction deposits.
Hypophysial Portal Infusion of GH-RF
17
(B ) Animals Infused with GH-RF Two separate and independent sources of a synthetic decapeptide with known growth hormone releasing activity were used (Nair GH-RF and Spectrum GH-RF). Because the morphological changes occurring after the infusion of either one of the GH-RFs were indistinguishable, the results will be presented collectively. All animals were infused for I min.
Fig. 3. Somatotrophs 1 min following 1-min infusion of GH-RF displayed emiocytic activity often with 2 or more granules coalescing and emerging through a common channel (long arrows). Empty vesicles are also seen at the cell membrane (short arrow). Glutaraldehyde fixation; stained with lead citrate and UA. x 20,000.
Downloaded by: Boston University 128.197.229.194 - 1/10/2019 11:50:03 PM
(!) 1-min Postinfusion Group Areas of multiple granule extrusion occurred I min following infusion of GH-RF (fig. 3). The RER was lamellar and polar as seen in controls. Granules remained dispersed throughout the cell with no specific alignment near the plasma membrane. Granules, when extruded into the perivascular space or basement membrane area, usually tended to be less dense, had less sharply defined margins, and appeared to show less of the limiting membrane. Acid phosphatase and TPPase activity was similar to infused controls.
18
W ilbur/W orthington/M arkwald
(2) 5-mirt Postinfusion Group The RER remained similar to that observed in controls. Many small smooth-surfaced and coated vesicles appeared in the proximity of the Golgi complex in close association with microtubules (fig. 4). Emiocytic activity continued to be extensive, showing many sites of multiple release. Occasional images were recorded of multiple granule cores appearing simultaneously within a plasmalemmal invagination opening into intercellular or pericapillary spaces. Granule number and distribution were similar to those observed in controls.
Fig. 4. Somatotrophs 5 min after 1-min infusion of GH-RF. Many small smooth surfaced and coated vesicles appeared near the Golgi complex in close association with microtubules (arrows). Glutaraldehyde-H,(); fixation; stained with lead citrate and UA. x 18,240.
Downloaded by: Boston University 128.197.229.194 - 1/10/2019 11:50:03 PM
(3) 15-min Postinfusion Group Golgi complexes appeared more hypertrophied than those from the pre vious groups. Microtubules near smooth-surfaced and coated vesicles were
Hypophysial Portal Infusion ofG H -RF
19
also seen near the Golgi complex (fig. 5). The RER was unchanged from controls and remained lamellar and diffuse. Although the granule content of individual somatotrophs varied, overall levels of granulation resembled those seen in controls. Coated invaginations were occasionally observed at plasma membranes. Emiocytic activity was present, although not as massive as in the 2 earlier time groups. Acid phosphatase activity was similar to control levels, as was TPPase activity.
Fig.5. Somatotroph 15 min following 1-min infusion of GH-RF. Numerous micro tubules (long arrows) and newly synthesized secretory granules (short arrows) are seen near the Golgi complex. Glutaraldehyde-H20 2 fixation; stained with lead citrate and UA. x 24,000.
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(4) 30-min Postinfusion Group Golgi complexes were vesicular and displayed elevated TPPase activity (fig. 6). Secretory granules continued to be observed within the Golgi com plex. The RER was distended and diffuse throughout the cell (fig. 7). Extreme variation in secretory granule number and distribution was evident on com-
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20 W ilbur/W orthington /M arkwald
Downloaded by: Boston University 128.197.229.194 - 1/10/2019 11:50:03 PM
Hypophysial Portal Infusion of GH-RF 21
22
W ilbur/W orthington /M arkwald
parison with earlier time groups. Emiocytic activity was less than that ob served at 1,5, and 15 min postinfusion. An increase in acid phosphatase ac tivity was observed in this time group. (5) 60-min Postinfusion Group Golgi complexes were similar to those observed in the previous 30-min group (fig. 8). The RER was not as distended as it was in the 30-min group, but it was more distended and diffuse than observed in controls. The number of secretory granules and their distribution were similar to observations of earlier time groups. Emiocytic activity was still present. An increase in the number of multivesicular and multilamellar bodies, along with acid phos phatase reactive lysosomes (fig. 9), was present in this group. TPPase activity was not elevated above control levels.
See Fig. 6-9, pages 20 and 21 Fig. 6. Somatotrophs 30 min following 1-min infusion of GH-RF continued to de monstrate distended and diffuse RER throughout the cell. Glutaraldehyde-H20 2 fixation; stained with lead citrate and UA. x 24,000. Fig. 7. Thiamine pyrophosphatase activity in somatotroph 30 min following 1-min infusion of GH-RF. Glutaraldehyde fixation; stained with UA only, x 38,000. Fig. 8. Somatotrophs 60 min following 1-min infusion of GH-RF. The Golgi complexes were vesicular and hypertrophied in somatotrophs containing few numbers of secretory granules. At this same time period in somatotrophs containing increased secretory granules, the Golgi complex resembled controls'. Glutaraldehyde-H20 2 fixation; stained with lead citrate and UA. x 24,000. Fig. 9. Acid phosphatase activity in a somatotroph 60 min following 1-min infusion of GH-RF. Gomori method. An increase in the number of acid phosphatase reactive granules (arrows) was observed at this time. Glutaraldehyde fixation; stained with UA only, x 48,000.
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(II) Radioimmunoassay o f Rat Growth Hormone (table II) A two-way analysis of variance followed by the calculation of the appro priate least significant differences [LSD (0.5) Appendix II] was used to deter mine if significant differences were present among the various treatment methods (dbcAMP vs GH-RFs: NGH-RF vs SGH-RF) and between preand postinfusion samples. (1) Following the 30-min infusion of dbcAMP, postinfusion levels of radioimmunoassayable plasma GH were significantly elevated (p
