Somatostatin receptor subtypes SSTR2 and SSTR5 couple negatively to an L-type Ca2+ current in the pituitary cell line AtT-20

Neuroscience Vol. 71, No. 4, pp. 1073 1081, 1996

~

Pergamon

0306-4522(95)00510-2

Elsevier ScienceLtd Copyright © 1996 IBRO Printed in Great Britain. All rights reserved 0306-4522/96 $15.00 + 0.00

S O M A T O S T A T I N R E C E P T O R S U B T Y P E S SSTR2 A N D SSTR5 C O U P L E N E G A T I V E L Y TO A N L-TYPE Ca 2+ C U R R E N T IN THE P I T U I T A R Y CELL LINE AtT-20 M. T A L L E N T , * G. L I A P A K I S , * A.-M. O ' C A R R O L L , t S. J. LOLAIT,'~ M. DICHTER:~ and T. R E I S I N E * § Departments of *Pharmacology and :~Neurology, University of Pennsylvania School of Medicine, 36th Street and Hamilton Walk, Philadelphia, PA 19104, U.S.A. tLaboratory of Cell Biology, National Institute of Mental Health, Bethesda, MD, 20892, U.S.A. Abstract--The somatostatin receptor subtypes SSTR2 and SSTR5 mediate distinct endocrine and exocrine functions of somatostatin and may also be involved in mediating the neuromodulatory actions of somatostatin in the brain. To investigate whether these receptors couple to voltage-sensitive Ca 2÷ channels, SSTR2 and SSTR5 selective agonists were tested for their effects on AtT-20 cells using whole cell patch clamp techniques. The SSTR2 selective agonist MK 678 inhibited Ca 2+ currents in AtT-20 cells. The effects of MK 678 were reversible and blocked by pertussis toxin pretreatment, suggesting that SSTR2 couples to the L-type Ca 2+ channels via G proteins. Other SSTR2-selective agonists, including BIM 23027 and NC8-12, were able to inhibit the Ca 2+ current in these cells. The SSTR5 selective agonist BIM 23052 also inhibited the Ca 2+ currents in these cells and this effect was reversible and blocked by pertussis toxin treatment. The ability of SSTR5 to mediate inhibition of the Ca ~+ current was greatly attenuated by pretreatment with the SSTR5-selective agonist BIM 23052, whereas SSTR2-mediated inhibition of the Ca 2÷ current was not altered by pretreatment with the SSTR2-selective agonist MK 678. Thus, the SSTR2 and SSTR5 couplings to the Ca 2+ current are differentially regulated. The peptide L362,855, which we previously have shown to have high affinity for the cloned SSTR5, had minimal effects on Ca 2+ currents in AtT-20 cells at concentrations up to 100 nM and did not alter the ability of MK 678 to inhibit Ca 2+ currents. However, it completely antagonized the effects of the SSTR5-selective agonist BIM 23052 on the Ca ~÷ currents. L362,855 is an antagonist/partial agonist at SSTR5 since it can reduce Ca 2+ currents in these cells at concentrations above 100 nM. L362,855 is also an antagonist/partial agonist at the cloned rat SSTR5 expressed in CHO cells since it is able to block the inhibition of cAMP accumulation induced by somatostatin at concentrations below 100 nM but at higher concentrations can inhibit cAMP formation itself. Structural analysis of L362,855 reveals that only a single hydroxyl group at residue seven in the peptide is needed to convert the compound from an antagonist/partial agonist to a full agonist at SSTR5. These studies reveal that two different somatostatin receptor subtypes, SSTR2 and SSTR5, can mediate the inhibition of an L-type Ca 2+ channel in AtT-20 cells by somatostatin. The receptor subtype responses can be distinguished by selective agonists and antagonists and are regulated differently by agonist pretreatment. The inhibition of Ca 2+ influx into endocrine cells and neurons may be a major cellular mechanism by which somatostatin modulates hormone and neurotransmitter release. Our results reveal that at least two receptor subtypes can mediate this cellular response. Key words: somatostatin, calcium current, neurotransmitter, somatostatin antagonist.

The peptide s o m a t o s t a t i n (SRIF) is a m a j o r inhibitor of hormone secretion from the endocrine system. 2~'7'13'14'17'2°'35'44'49S R I F is able to block growth h o r m o n e release from the anterior pituitary and glucagon and insulin secretion f r o m the pancreas. This peptide also acts as a n e u r o m o d u l a t o r a n d / o r n e u r o t r a n s m i t t e r in the brain, where it inhibits §To whom correspondence should be addressed. Abbreviations : DMEM, Dulbecco's modified Eagle's medium; EGTA, ethyleneglycol-bis(/3-aminoethyl ether)-N,N,N',N'-tetra-acetate; GTP, guanine triphosphate; HEPES, N-(2-hydroxyethyl) piperazine-N'-yl (2-ethanesulphonic acid); IBMX, isobutylmethylxanthanine; /Ca, calcium current; PTX, pertussis toxin; SRIF, somatostatin; SRIF 28, somatostatin 28; TEA, tetraethylammonium; TTX, tetrodotoxin. Nsc 71/4~3

neuronal activity and modulates n e u r o t r a n s m i t t e r release and may have a role in regulating m o t o r and cognitive f u n c t i o n s ) ~14'19'47 Multiple cellular actions are m o d u l a t e d by SRIF. S R I F blocks adenylyl cyclase activity 23'34'47'5°'58 and has recently been s h o w n to stimulate tyrosine phosphatase activity to inhibit cell g r o w t h ) °'11'32'42 In neurons and endocrine cells, S R I F potentiates K ÷ currents to hyperpolarize cells and reduce firing r a t e . 37'38'6°'61'63'67 S R I F also reduces Ca 2+ currents in neurons and secretory cells, 16'22'24"31'33'36'57'62 which has been linked to its ability to inhibit h o r m o n e and n e u r o t r a n s m i t t e r secretion. 33'53 A family o f five S R I F receptor subtypes has recently been c l o n e d . 1'5'8'9'21'39'4°,6*-66 Subtype selective agonists have been identified that interact with

1073

M. Tallent et al.

1074

S S T R 2 a n d S S T R 5 . 21'45'46'5'~56 These c o m p o u n d s have been useful in delineating specific functions of these receptor subtypes. SSTR2 has been p r o p o s e d to be involved in S R I F - m e d i a t e d inhibition of g r o w t h h o r m o n e release from the pituitary, 45'46 glucagon secretion f r o m the p a n c r e a s 55 a n d gastric acid secretion from the stomach. 56 SSTR5 has been postulated to be involved in the inhibition o f insulin secretion by SRIF. 54 These results suggest t h a t S R I F receptor subtypes m a y mediate distinct functions of SRIF. A t a cellular level, b o t h SSTR2 a n d SSTR5 couple to adenylyl cyclase to mediate the inhibition o f c A M P a c c u m u l a t i o n by S R I F . 21'39'4°'52SSTR2 also couples to a tyrosine p h o s p h a t a s e t°,H a n d stimulation of this enzyme has been linked to the ability of S a n d o s t a t i n 6 a n d o t h e r clinically used S R I F analogs to inhibit t u m o r cell growth. 21'25'26 SSTR5 does n o t a p p e a r to couple to this tyrosine p h o s p h a t a s e b u t has been r e p o r t e d to decrease Ca 2+ mobilization H to inhibit t u m o r cell growth. There is little i n f o r m a t i o n regarding the electrophysiological responses regulated by S R I F receptor subtypes which m a y be m o s t critical in evaluating the n e u r o t r a n s m i t t e r a n d n e u r o r n o d u latory actions of SRIF. A model cell system t h a t has been extensively used to study the electrophysiological actions of S R I F is the cell line AtT-20, which is derived from the m o u s e a n t e r i o r pituitary. 31'33'34'5°'51'53'58These cells express an L-type Ca 2÷ c h a n n e l a n d S R I F has been reported to reduce Ca 2÷ c o n d u c t a n c e in these cells to block h o r m o n e secretion. 3L33 F u r t h e r m o r e , these cells express b o t h S S T R 2 a n d SSTR5 m R N A . 39'43 In the present study we used SSTR2 a n d SSTR5 selective ligands to test w h e t h e r these receptor subtypes couple to v o l t a g e - d e p e n d e n t Ca 2÷ c h a n n e l s in ART-20 cells. EXPERIMENTAL PROCEDURES

Tissue culture

Chinese hamster ovary (CHO-K l) cells expressing rat and human SSTR5 were grown in Hams's F-12 medium with 10% fetal bovine serum as previously described. 39"4°'46AtT20 cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum and antibiotics as previously described. 27,3~'33'34,5°,5~ Peptides

The peptides c[Phe-Trp-D-Trp-Lys-Thr-Phe-AHA] (L362,855) (AHA refers to 7-amino heptanoic acid), c[TyrTrp-D-Trp-Lys-Thr-Phe-AHA] (L-372,587) and c[Phe-Trp-D-Trp-Lys-Thr-Tyr-AHA] (L-372,588) were synthesized and provided by Dr Roger Friedinger at Merck (Rahway, N J). MK 678 was also provided by Merck and BIM 23027, BIM 23052 and NC8-12 were provided by Biomeasure (Milford, MA). SRIF was purchased from Bachem (Torrence, CA). c A M P accumulation

Accumulation of cAMP in cells expressing SSTR5 was measured as previously described. ~,52 Briefly, cells cultured in 12-well Costar plates were incubated with medium containing 10% fetal bovine serum and 500/~M isobutylmethylxanthanine (IBMX) for 10min. The medium was removed and replaced with similar medium containing

10 # M forskolin in the presence or absence of various SRIF analogs. After incubation for 30 min at 37°C in 10% CO2, the medium was removed, 0.5 ml of 1 N HC1 was added to the cells and the cells were sonicated. A 5-/t 1 aliquot of each sample was then analysed by radioimmune assay to measure cAMP content using a commercially available kit (NEN/DuPont, Boston, MA). Electrophysiological studies

AtT-20 cells were plated at a density of 1 × 105 cells in 35mm 2 dishes 3-7 days before use. The whole-cell version of the patch clamp technique ~8was used for recording ionic currents using an Axopatch 200A amplifier (Axon Instruments) with a PDPll/73 computer interface (Indec Systems). For determination of peak current to voltage relationships, cells were held at - 8 0 mV and the voltage was stepped in 10mV intervals from - 5 0 to +50 for 15 ms with 2 s between steps. Leak currents were subtracted from all records and currents were filtered at 5 kHz. Test drugs were dissolved in external solution and applied to cells via pressure ejection from multi-barreled glass pipettes with a tip diameter of 5 10 # m held 30-50 # m from the cell. The external solution contained 140mM NaC1, 1 mM KC1, l mM MgC12, 10mM CaCl2, 5 m M glucose, 10mM HEPES, 5 # M tetrodotoxin (TTX) and 2 mM 4-aminopyridine, at a pH of 7.3. The pipette solution consisted of 120 mM CsCl, 11 mM EGTA, 2 mM tetraethylammonium (TEA) 2 mM MgC12, 10 mM HEPES and 4 mM ATP and 200 # M guanine triphosphate (GTP) at a pH of 7.3. Patch pipettes had a tip resistance of 3-5 Mr2 when filled. All recordings were done at room temperature. Desensitization studies

AtT-20 cells were incubated for 1 h in medium containing either 1 # M MK 678 or 1/tM BIM 23052. The medium was removed, the cells were washed three times with fresh external solution without peptides, and recordings were conducted in fresh external solution. Recordings were conducted within 30 min after removal of drug. RESULTS

SSTR2

couples to an L - t y p e Ca 2+ current

Previous studies have s h o w n t h a t AtT-20 cells express S R I F receptors 27,34"5°'5j'53'58a n d t h a t S R I F can reduce L-type C a 2+ currents 3~ in these cells. To identify the type o f Ca z+ currents present in AtT-20 cells, we recorded currents in the presence of 10 a n d 50/~M nimodipine, a dihydropyridine which is a selective L-type Ca 2+ c h a n n e l blocker. In the absence of nimodipine, the peak average C a 2+ current in nine cells was 164_+ 11 pA. Average peak Ca 2+ currents recorded in 1 0 # M n i m o d i p i n e were 47.2_+ 1 0 p A (n = 5) a n d in 5 0 # M n i m o d i p i n e were 23.8 _+ 21 p A (n = 5), representing a n inhibition of 71 a n d 85%, respectively. N o inhibition of the Ca 2+ currents was seen u p o n application of 2/~ M o m e g a - c o n o t o x i n , an N-type Ca 2+ channel antagonist. Therefore, the majority o f Ca 2+ currents in AtT-20 cells a p p e a r e d to be mediated by L-type Ca 2+ channels. Previous studies have s h o w n t h a t AtT-20 cells express SSTR2 m R N A , 43 SSTR2-1ike i m m u n o r e a c tivity 43'59 a n d receptors with high affinity for SSTR2 agonists such as M K 678. 27 The affinity 0c50 value) of AtT-20 cell S R I F receptors for M K 678, as m e a s u r e d by its ability to inhibit 125I-MK 678 binding, is 0.23 nM. M K 678 (100 n M ) was able to inhibit the

SSTR2 and SSTR5 couple to Ca 2+ currents Ca 2÷ currents in AtT-20 cells, as seen in Fig. 1, to a similar extent as seen with 10 nM S R I F (see Table 1). Shown in Fig. I A are current traces from a representative cell in which 100 n M M K 678 reversibly inhibits the calcium current (Ica) from 2 4 5 p A to 160pA, representing a 35% inhibition. Figure 1B shows a typical current to voltage ( l - V ) plot for a representative cell. The Ca 2÷ current begins to activate at - 3 0 m V and peaks at + 2 0 m V . M K 678 inhibition does not alter the voltage dependency of the current. Inhibition of the Ca 2÷ current by M K 678 was concentration dependent, and the ECs0 for M K 678 inhibition was 1.3 n M (Fig. 2). Modulation of the L-type Ca 2÷ current by M K 678 is sensitive to pertussis toxin (PTX), as overnight treatment with 100 ng/ml PTX abolishes the inhibition by M K 678 (Table 1). This indicates that SSTR2-1ike receptors in AtT-20 cells couple to the Ca 2÷ current through a G protein-mediated mechanism. While M K 678 can interact with other S R I F receptor subtypes besides SSTR2 at high concentrations, BIM 23027 and NC8-12 selectively interact with SSTR2. 4°'45'46 The affinity of BIM 23027 for

1075

AtT-20 cell S R I F receptors as measured by its ability to inhibit [125I]MK 678 binding is 0.001 nM. BIM 23027 (100 nM) and NC8-12 (10 nM) each inhibited the Ca 2÷ current by 21.3 __+2 and 32.0 _ 7%, respectively (Table 1). The ability of the SSTR2-selective agonists to inhibit the Ca 2+ current suggests that SSTR2 is expressed in AtT-20 cells and that it functionally couples to a Ca ~+ current. S S T R 5 couples to an L - t y p e Ca 2+ current

Previous studies have shown that SSTR5 m R N A is also expressed in AtT-20 cells, 39 suggesting that this receptor subtype may also be present in these cells. The peptide BIM 23052 has an affinity (Ics0 value) for AtT-20 cell S R I F receptors, as measured by its ability to inhibit [125I]CGP 23996 binding, of 0.031 nM. The SSTR5-selective agonist BIM 23052 inhibited the L-type Ca 2÷ current in these ceils. Shown in Fig. 3A are current traces from a representative cell in which 100 nM BIM 23052 reversibly inhibits the Ca 2÷ current. An I- V curve from a representative cell is shown in Fig. 3B. The average reduction in Ic~ by 100 nM BIM 23052 was 2 5 . 6 _ 2 % (Table 1). BIM 23052

A MK678

2.5 ms

washes

V

voltage (mY)

B

-60

-40

-20

0

20

40

60

Fig. 1. MK 678 (100 nM) inhibits/ca in AtT-20 cells. (A) Current traces from a representative cell elicited from a holding potential from - 8 0 mV by stepping to +20 mV for 15 ms. (B) Peak current to voltage ( l - V ) plot for a representative cell. Ca z+ currents began to activate at - 3 0 mV and peaked at + 20 mV. MK 678 inhibition did not change the voltage-dependent characteristics of the current.

M. Tallent et al.

1076 100

r~

c ffl ~

60

"~

40

Antagonistic effects of L362,855

20 0 0.0

indicate t h a t M K 678 a n d B I M 23052 selectively interact with different receptors to inhibit Ca 2+ currents in ART-20 cells. F u r t h e r m o r e , these results show t h a t SSTR2 a n d SSTR5 responses in AtT-20 cells can be distinguished by their differential sensitivity to agonist-induced desensitization.

~ 6 , , . 0.1

I

I

I

I

10

100

concentration M K 678 (nM)

Fig. 2. MK 678 inhibition of ~co is dose dependent. Per cent of maximal inhibition of/Ca is plotted as a function of MK 678 concentration. Data were fitted to the Hill equation (Y = b0 - (b0/1 + X/b2)bl, where b 0 is the maximal response, b t is the Hill coefficient and b2 is the Ecs0 value. For this curve fit, b0 = 97.8, b~ = 1.01, and b z = 1.3. Results are the mean _+ S.E.M. of at least three different experiments.

( 1 0 n M ) was able to inhibit the Ca 2+ current by 20.3 + 2 % (Table 1). The inhibition by 100 n M B I M 23052 was blocked by overnight t r e a t m e n t with P T X (100 ng/ml), indicating t h a t SSTR5 is coupled to the Ca 2+ current via a G protein (Table 1).

Selective desensitization of S S T R 5 Previous studies have s h o w n t h a t the coupling o f a SSTR2-1ike receptor to Ca 2+ channels in cultured cortical n e u r o n s does not desensitize following prolonged agonist pretreatment. 48'62 Similarly, pretreatm e n t of AtT-20 cells with 1 # M M K 678 for 1 h did n o t reduce the subsequent ability o f M K 678 to inhibit the L-type Ca 2+ current. The inhibition by 100 n M M K 678 after p r e t r e a t m e n t with 1 # M M K 678 was 27.0 + 0.7% (n = 4) which was n o t different from u n t r e a t e d control cells (27 ___3%). In the same g r o u p of cells pretreated with 1 / t M M K 678, 100 n M B I M 23052 inhibited the Ca 2÷ c u r r e n t by 26.1 + 4 % (n = 4), which is the same as the inhibition by B I M 23052 in n o n t r e a t e d cells (26 +- 2%). P r e t r e a t m e n t of AtT-20 cells for 5 h with 1 # M M K 678 also did n o t diminish the ability of M K 678 to inhibit the C a 2+ current (percentage inhibition of the Ca 2÷ c u r r e n t by 100 n M M K 678 was 29 + 2 % , n = 3), further indicating t h a t SSTR2 coupling to the Ca 2+ c h a n n e l is resistant to desensitization. In c o n t r a s t to the lack of effects of M K 678 p r e t r e a t m e n t , p r e t r e a t m e n t of AtT-20 cells for 1 h with 1 # M B I M 23052 selectively desensitized SSTR5. B I M 23052 ( 1 0 0 n M ) inhibited /ca by only 1.3 + 0.9% (n = 7) after pretreatment, c o m p a r e d to 21.3 +- 2.7% (n = 8) in control cells. P r e t r e a t m e n t with 1 # M B I M 23052 did n o t alter the ability of M K 678 to inhibit Ic,, as the inhibition in control cells was 25.5 + 2.6% (n = 8), a n d in cells pretreated with B I M 23052 was 22.4 +- 0.8% (n = 7). These findings

F u r t h e r evidence t h a t distinct receptor subtypes mediate the inhibition of C a 2+ c o n d u c t a n c e in AtT20 cells by S R I F is provided by studies with the peptide L362,855. 46 This peptide exhibits high affinity for the cloned SSTR5, h a v i n g similar potency to B I M 23052. 46 This peptide has an affinity at AtT-20 cell S R I F receptors o f 0.003 n M , as m e a s u r e d by its ability to inhibit [12sI]CGP 23996 binding. Unlike B I M 23052, L362,855 was u n a b l e to inhibit the Ca 2+ current, with 100 n M h a v i n g no effect. A t a concent r a t i o n of I # M , L362,855 caused a 15.2+_4% inhibition o f the current (Table 1). This inhibition was significantly less t h a n the maximal inhibition of B I M 23052, consistent with the partial agonist effect o f L362,855 at very high c o n c e n t r a t i o n s (1000-fold higher t h a n its affinity at binding to SSTR5). However, L362,855 a n t a g o n i z e d the effects of B I M 23052, since in the presence of 100 n M L362,855, B I M 23052 was no longer able to inhibit the Ca 2+ current (Table 1, Fig. 4A). The effects of L362,855 appeared to be selective for SSTR5, since the SSTR2-selective agonist M K 678 inhibited the Ca 2+ current to a similar m a x i m a l effect in the presence (27.0 +- 3%) a n d absence ( 2 7 . 3 + - 3 % ) of 1 0 0 n M L362,855 Table 1. Inhibition of the L-type Ca 2+ current in AtT-20 cells by somatostatin analogs Ligand SRIF (10nM) SSTR5-selective BIM23052 (100 nM) BIM23052 (100 nM) + L362,855 (100 nM) BIM23052 (100 nM) + P T X (100 ng/ml) BIM23052 (10 nM) BIM23052 (10 nM) + L362,855 (100 nM) L362,855 (100 nM) L362,855 (1000 nM) L372,588 (100 nM) L372,588 (100 nM) +L372,587 (100 nM) L372,587 (100 nM) L372,587 (1000 riM) SSTR2-selective BIM23027 (100 nM) NC8-12 (10 nM) MK 678 (100 nM) MK 678 (100 nM) +L362,855 (100 nM) MK 678 (100 nM) + P T X (100 ng/ml)

% Mean inhibition _+ S.E.M. of the Ca 2+ current

n

31.0_+ 3

3

25.6 + 2

6

6.4 + 2

6

2+ 1 20.3 + 2

6

3.9 _+ 2 0.5 + 0.5 15.2 _ 4 27.2 _+2

6 8 6 5

4.0 _+2 3.3 + 2 1.4 _+ 1

5 8 6

21.3 + 2 32.0 + 7 27.3 + 3

4 4 7

27.0 + 3

7

1+ 1

3

6

Values shown are the percentage inhibition of the peak Ca 2+ current elicited by a voltage step from -- 80 to + 20 mV.

SSTR2 and SSTR5 couple to (Table 1, Fig. 4B). These studies further establish that the inhibition of Ca 2÷ currents in AtT-20 cells by SRIF is independently mediated by SSTR2 and SSTR5, and suggest that L362,855 is an antagonist on SSTR5. To investigate further whether L362,855 acts as an antagonist/partial agonist on SSTR5, the ability of the peptide to inhibit forskolin-stimulated cAMP accumulation in CHO-K1 cells expressing rat SSTR5 was tested. At concentrations up to 1/~M, L362,855 maximally inhibited forskolin-stimulated cAMP accumulation by 28 + 7 % . In contrast, 1/~M SRIF caused an 89 + 4% reduction in forskolin-stimulated cAMP accumulation in SSTR5 expressing CHO cells (n = 4). The addition of 100 nM L362,855 and 1/~M SRIF resulted in only a 3 6 + 10% inhibition of cAMP formation, indicating that L362,855 blocked inhibition of cAMP accumulation induced by SRIF. The antagonism of SRIF's actions by L362,855 was concentration dependent (Fig. 5). The blockade was also observed in CHO cells expressing human SSTR5 (% Inhibition of forskolin-stimulated cAMP accumulation, I # M SRIF = 88 _+4% ; 1/~M L362,855 = 34_+ 7% ; 1 ktM SRIF + 1 # M L362,855 = 31 _+ 6%).

C a 2+

currents

1077

L362,855 has two phenylalanine residues in its structure. Conversion of one of the phenylalanines at position 7 to a tyrosine generated the peptide L372,588. This peptide is a full agonist at SSTR5 since 100nM L372,588 inhibits the Ca 2+ current in AtT-20 cells by 2 7 . 2 _ 2%, which is similar to the level observed with BIM 23052 (Table 1, Fig. 4C). Furthermore, L372,588 was able to inhibit forskolin-stimulated cAMP accumulation in CHO cells expressing rat SSTR5 to the same extent as SRIF, and when co-applied with SRIF did not diminish the inhibitory effects of SRIF (not shown). In contrast, L372,587, with the phenylalanine at position 2 converted to a tyrosine, acted in a similar manner to L362,855. L372,587 had no effect on the Ca 2+ currents in AtT-20 cells at 100 nM and 1 #M (Table 1, Fig. 4D) but was able to antagonize fully the ability of L372,588 to inhibit the Ca 2÷ current (Table 1, Fig. 4C). These findings indicate that a single hydroxyl group at position 7 of L362,855 is critical for the agonistic properties of this peptide and that removal of this hydroxyl group generates an antagonist/partial agonist at SSTR5.

A

washes 2.5 ms

VH=-80mV VSTEP= +20 mV

B voltage (mV) -60

-40

-20

0

20

|

O Z~ []

|

control BIM 23052 wash

40 |

60 |

-200

-300 --1 O

-400

Fig. 3. The SSTR5-selective ligand BIM 23052 inhibits /ca in AtT-20 cells. (A) Current traces from a representative cell elicited from a holding potential from - 8 0 mV by stepping to + 20 mV for 15 sec. (B) I - V curve from a representative cell: 100 nM BIM 23052 is shown to inhibit reversibly the current without changing the voltage-dependent characteristics of the current.

M. Tallent et al.

1078

A

BIM~3052

B

MK 678/MK 678 + L362,855

¢--wash/BIM 23052 + L362,855

wash < o

2.5 ms

C

L372,588

D

wastVL372,587

wash/L372,588 + L372,587

VH= -80 mV Vstep= +20 mV

Fig. 4. Current traces from representative cells showing modulation of Ic, by L362,855 and its analogs. (A) BIM 23052 (100 nM) inhibits Ic, in this cell. Co-application of (100 nM) L362,855 blocks the ability of 100nM BIM 23052 to inhibit /ca- (B) MK 678 100nM is able to inhibit the current in this cell. Co-application of 100 nM L362,855 does not alter the ability of 100 nM MK 678 to inhibit Ic,. (C) L372,588, a derivative of L362,855 with a phenylalanine to tyrosine substitution at position 7, is able significantly to inhibit /Ca at 100 nM. In contrast, the compound L372,587 (phenylalanine to tyrosine substitution at position 2) has antagonistic properties, in that it blocks the inhibition by 100 nM L372,588 when co-applied at 100 nM, a concentration at which it has no effect of its own (D).

DISCUSSION The two receptor subtypes SSTR2 a n d SSTR5 have been s h o w n to mediate inhibition of h o r m o n e secretion f r o m endocrine a n d exocrine organs by S R I F . SSTR2 has been p r o p o s e d to be involved in the c o n t r o l o f g r o w t h h o r m o n e release from the a n t e r i o r pituitary in vitro. 45'46 U s i n g the subtype selective agonist NC8-12, Rossowski a n d Coy 55'56 suggested t h a t SSTR2 is also selectively involved in the regulation o f glucagon a n d gastric acid secretion by S R I F in vivo. These same a u t h o r s 54'56 used the selective agonist B I M 23052 to indicate t h a t SSTR5 mediates the regulation o f insulin a n d amylase secretion in vivo by SRIF. T h e ability of S R I F to inhibit h o r m o n e secretion has been linked to its ability to inhibit C a 2+ c o n d u c t a n c e a n d Ca 2+ influx into secretory cells. 31'33'53T h e present study shows t h a t b o t h S S T R 2 a n d SSTR5 can couple negatively to a n L-type Ca 2+ c h a n n e l to reduce C a 2+ influx. Recent studies by Fujii et a l J 6 revealed t h a t the cloned rat S S T R 2 expressed in R I N cells can couple to voltage-sensitive Ca 2+ c h a n n e l s to mediate inhibition o f C a 2÷ influx by SRIF. O u r studies show t h a t SSTR2 e n d o g e n o u s l y expressed in AtT-20 cells couples to a n L-type C a 2+ channel. Structurally diverse SSTR2-selective agonists inhibited the current a n d their effects were blocked by pertussis toxin treatment, suggesting t h a t SSTR2 couples to the C a 2+ c h a n n e l via G proteins. Kleuss et al. 24 have reported t h a t S R I F receptors couple to C a 2+ c h a n nels via Goa in GH3 cells. Previous studies by Law et al. 27 30 have s h o w n t h a t Go. physically associates

with S R I F receptors in AtT-20 cells t h a t have high affinity for M K 678 a n d the cloned SSTR2 expressed in C H O - D G 4 4 cells. T o g e t h e r these findings suggest t h a t SSTR2 couples to Ca 2÷ channels via Goa. LL

~:IOO O3 c

.~

8C

~ ~o ~

40

r, 20

O.Ol

I o.1

I 1

I lO

I lOO

concentration L362,855 (nM)

Fig. 5. L362,855 blocks the inhibition of cAMP accumulation by SRIF in CHO ceils expressing rat SSTR5. CHO ceils expressing rat SSTR5 were treated with 10pM forskolin alone or in the presence of 1000 nM SRIF alone or in the presence of varying concentrations of L362,855. cAMP content in the cells was measured as described in the Experimental Procedures. Basal cAMP levels, which measured less than 10% of the forskolin-stimulated values, have been subtracted from the stimulated values and percentage inhibition values determined. At a concentration of 1000 nM, L362,855 inhibited forskolin-stimulated cAMP accumulation by 28 + 7%. These are the results of at least three different experiments. Data were fitted to the Hill equation. The calculated Ic50 value is 6.3 nM.

SSTR2 and SSTR5 couple to Ca2+ currents The coupling of SSTR2 to Ca 2÷ channels was maintained following prolonged agonist pretreatment, indicating that SSTR2/Ca 2÷ channel coupling did not desensitize. Similar results were obtained previously with SRIF receptors expressed in embryonic rat cortical neurons in culture that have high affinity for M K 678. 62 In those cells, SRIF pretreatment did not desensitize the coupling of the SRIF receptors linked to Ca 2+ channels. The resistance of SSTR2/Ca 2÷ channel coupling to desensitization differs from the coupling of SSTR2 to adenylyl cyclase, which rapidly desensitizes.52 Differential regulation of cellular responses linked to SSTR2 may be due to their distinct coupling mechanisms, since SSTR2 has been proposed to couple to adenylyl cyclase via Gia29 but couples to Ca 2÷ channels via Goa. SSTR5 has also been reported to mediate effects of SRIF on hormone release, s4 Its inhibitory activity on secretion may be associated with its ability to inhibit Ca 2÷ influx into cells. Like SSTR2, SSTR5 coupling to Ca 2+ channels is PTX sensitive, suggesting that G proteins link the receptor to the Ca 2+ channels. However, unlike SSTR2 responses, the coupling of SSTR5 to Ca 2÷ channels desensitizes. Previous studies have also shown that coupling of the cloned rat SSTR5 to adenylyl cyclase in CHO cells desensitizes following prolonged agonist treatment. 46 The differential regulation of SSTR2 and SSTR5 coupling to Ca 2+ channels in AtT-20 cells suggests either that these receptors have distinct cellular mechanisms linking them to L-type Ca 2+ channels, or that the molecular events involved in their regulation differ. MK 678 was able fully to inhibit Ca 2+ currents in AtT-20 cells desensitized to BIM 23052. Furthermore, in cells chronically treated with MK 678, BIM 23052 fully inhibited C a 2+ currents, indicating that M K 678 did not desensitize SSTR5. MK 678 has been reported to interact with rat SSTR5 with moderate affinity.2~'46Its inability to desensitize SSTR5 suggests that it may not be a full agonist at this receptor. In fact, recent studies by Feniuk et al. 15 have shown that MK 678 has partial agonist/antagonist properties at a receptor expressed in guinea-pig atrium. This receptor has higher affinity for SRIF 28 than SRIF, which is a unique characteristic of SSTR5. The coupling of SSTR2 and SSTR5 to Ca 2+ channels in AtT-20 cells was also distinguished by the inhibition produced by L362,855. This peptide has high affinity for rat and human SSTR5. 4° However, it did not inhibit Ca 2+ currents in ART-20 cells at 100 nM and had minimal effects on forskolin-stimulated cAMP accumulation in CHO cells expressing the cloned rat SSTR5. At this concentration, L362,855 did block SSTR5-mediated responses, both in the AtT-20 cells and CHO cells. L362,855 selectively blocked SSTR5-mediated electrophysiological responses since it did not alter the ability of M K 678 to inhibit Ca 2÷ currents in AtT-20 cells. L362,855 inhibits Ca 2+ currents at concentrations above 100 nM. This effect could be due to a selective

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stimulation of SSTR5. The level of inhibition of the Ca 2÷ current induced by 1/~M L362,855 was less than that seen with BIM 23052 or SRIF, suggesting that L362,855 may be a partial agonist. Alternatively, at these high concentrations, L362,855 may be acting on another SRIF receptor subtype. The antagonism of the effects of BIM 23052 on Ca 2+ currents occurred at lower concentrations and was selective, since L362,855 did not block the actions of MK 678. While L362,855 is not a pure SSTR5 antagonist, it may serve as the basis for the development of a new generation of SSTR5 ligands. The addition of a hydroxyl group at position 7 of L362,855 resulted in L372,588 (conversion of a phenylalanine to a tyrosine), which is a pure agonist at SSTR5. L372,588 inhibited Ca 2+ currents in AtT-20 cells to the same extent as BIM 23052, and reduced forskolin-stimulated cAMP accumulation in cloned SSTR5 expressing cells to the same extent as SRIF. Furthermore, it did not block the actions of SRIF at SSTR5. The ability of a simple hydroxyl group to confer pure agonism versus antagonism reveals an important region of L362,855 critical for the intrinsic activity of the peptide. Since SSTR5 selective agonists have been reported to inhibit insulin release, 54implying that this receptor is involved in the control of insulin release, the development of SSTR5-selective antagonists could be useful in the treatment of individuals with hypoinsulin secretion, such as those with diabetes. The role of single hydroxyl groups in ligand binding to SSTR5 has recently been established in mutagenesis studies on the cloned rat SSTR5. Ozenberger and Hadcock 41 showed that conversion of a phenylalanine at residue 265 in SSTR5 to a tyrosine greatly increases the affinity of the receptor for SRIF, such that SRIF and SRIF 28 have similar affinity at this receptor. Hydroxyl groups may be important for electrostatic interactions of SRIF peptides with their receptors. Identification of which hydroxyl groups in the receptor and peptide are needed for the highaffinity binding and for intrinsic activity should facilitate rational design of new SSTR5 ligands. CONCLUSION

In conclusion, we show that SSTR2 and SSTR5, which may have important roles in mediating the inhibitory actions of SRIF on hormone secretion, both couple to L-type Ca 2+ channels in AtT-20 cells. The inhibition of Ca 2+ conductance mediated by these receptors may be critical in their antisecretory functions. We also show that the peptide L362,855 has antagonistic properties at SSTR5, and that the phenylalanine at position 7 is critical for its antagonistic properties. This information may be useful in the development of better SSTR5-selective antagonists. A c k n o w l e d g e m e n t s - - W e thank Dr Roger Friedinger for

providing us with L362,855, L372,588 and L372,587. This work was supported by NIMH grants MH45533 and MH48518.

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